|Year : 2020 | Volume
| Issue : 5 | Page : 535-540
Comparison of oocyte fertilization rates using different culture systems: A prospective cohort study
Sandeep Karunakaran, Uttara Aiyer Kohli
Department of Obstetrics and Gynaecology, Command Hospital Air Force, Bengaluru, Karnataka, India
|Date of Submission||24-Sep-2019|
|Date of Decision||12-Dec-2019|
|Date of Acceptance||03-Mar-2020|
|Date of Web Publication||7-Sep-2020|
Uttara Aiyer Kohli
Department of Obstetrics and Gynaecology, Command Hospital Air Force, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Context: The success of clinicalin vitro fertilization (IVF) may be hampered by suboptimal oocyte culture. Research over the past 25 years has resulted in the development of more physiological and effective culture systems capable of maintaining both development and viability of the preimplantation embryo, resulting in the increase in the overall success rates of human-assisted conception per cycle. However, the perfect culture system has not yet been determined.Aims: The aim of the study was to compare the difference in the fertilization rates of oocytes using two different culture systems – cohort co-culture and microdroplet culture methods.Settings and Design: This was an observational, prospective cohort study carried out in a tertiary care hospital with an assisted reproductive center between June 2017 and August 2018.Materials and Methods: Oocytes from normoresponsive patients undergoing IVF and meeting the inclusion and exclusion criteria were divided into two groups and cultured using either the cohort (group) or the microdroplet culture methods using conventional IVF.Statistical Analysis Used: Details of all cases were recorded on a structured format and analyzed with the help of registered version of SPSS version 21 using the Chi-square test and Student's t-test. Statistical significance was assessed at P < 0.05.Results: Three hundred and twenty-seven patients (159 [1031 oocytes] in cohort and 168 [1067 oocytes] in microdroplet culture) were included in the study. No significant difference (P = 0.08) was found in the fertilization rates in the two culture systems; however, denudation of oocyte was faster in the microdroplet group.Conclusions: The difference in the fertilization rates of oocytes using cohort culture or microdroplet culture systems was not statistically significant.
Keywords: Co-culture, in vitro fertilization, microdroplet culture, oocyte culture
|How to cite this article:|
Karunakaran S, Kohli UA. Comparison of oocyte fertilization rates using different culture systems: A prospective cohort study. Med J DY Patil Vidyapeeth 2020;13:535-40
|How to cite this URL:|
Karunakaran S, Kohli UA. Comparison of oocyte fertilization rates using different culture systems: A prospective cohort study. Med J DY Patil Vidyapeeth [serial online] 2020 [cited 2020 Sep 20];13:535-40. Available from: http://www.mjdrdypv.org/text.asp?2020/13/5/535/294355
| Introduction|| |
The initial success of clinicalin vitro fertilization (IVF) was compromised by suboptimal culture conditions, resulting in impaired embryo development, and all too frequently complete developmental arrest around the 8-cell stage.,,, Consequently, it became the paradigm to transfer human embryos to the uterus asynchronously on day 1, 2, or 3. It was advocated that if the laboratory conditions were not optimized, then embryos should be transferred as soon as possible back to the uterus to avoid suboptimal conditions.
Fortunately, research over the past 25 years has resulted in the development of more physiological and effective culture systems capable of maintaining both development and viability of the preimplantation embryo, culminating in the routine culture of human blastocysts.,, Improvements in embryo culture media formulations, combined with increases in efficiency and safety of the overall culture system, have led directly to a significant increase in embryo implantation rates (for both cleavage and blastocyst transfers), and a decline in pregnancy loss, thereby facilitating the routine introduction of single embryo transfer. Furthermore, more suitable culture conditions produce embryos more able to survive cryopreservation.
Improvements in embryo culture technology have significantly contributed to the increase in the overall success rates of human-assisted conception per cycle. However, the perfect culture system has not yet been determined. There has been a thought process that the presence of embryos being co-cultured might help the process of fertilization and their further development by the presence of autocrine and paracrine factors. This scientific thought also has not reached its conclusion.
The aim of the study was to compare the difference in the fertilization rates of oocytes using two different culture systems – cohort co-culture and microdroplet culture methods.
We also studied the time taken to denude the inseminated oocytes in the two different culture systems as a secondary outcome.
| Materials and Methods|| |
This was an observational, prospective cohort study carried out in a tertiary care hospital having an assisted reproductive center between June 2017 and August 2018, to study the question of whether co-culture helps in improving the fertilization rates. Ethical clearance was taken from the Institutional ethics committee on August 02, 2016. Consent was taken from the participants to use data.
Oocytes from normoresponsive patients undergoing IVF were divided into two groups and cultured using either the cohort (group) or the microdroplet culture methods using conventional IVF.
All patients undergoing IVF between the age groups of 21 and 35 years were included in the study.
The following patients were excluded from the study:
- Poor ovarian response (<3 oocytes retrieved from ovum pick-up [OPU])
- O!varian hyper-response (>20 oocytes retrieved from OPU)
- Polycystic ovarian syndrome
- History of total fertilization failure in previous IVF cycle
- Gross disparity between the observed follicle number and the number of follicles retrieved
- Total fertilization failure in the present IVF cycle
- Postwash semen concentration ɢ million/ml
- Cases that required intracytoplasmic sperm injection.
Sample size calculation
Since a similar study was not done in the past, a reasonable sample size was determined to be 1000 oocytes in each arm, namely the cohort (group) culture group and the microdroplet culture group.
The patients underwent controlled ovarian hyperstimulation using the long luteal agonist protocol. The agonist used was injection triptorelin. It was started from the 21st day of the preceding cycle and continued till the day of human chorionic gonadotropin (hCG) trigger. The patients were stimulated from day 2 of their cycle with injection recombinant follicular stimulating hormone of 150–225 IU. The patients were monitored by transvaginal sonography (TVS), and when the follicle size of the leading cohort reached between 18 and 20 mm, the final maturation was triggered using injection hCG of 10,000 IU. The number of follicles was noted on the day of the hCG trigger.
About 34–36 h after the administration of injection hCG, the patients were scheduled for OPU which was carried out under total intravenous anesthesia. A 17G oocyte retrieval needle was used, and the returning follicular fluid was collected in 14-ml round-bottom test tubes at 37°C. The retrieved follicular fluid was then examined for the presence of oocyte-cumulus complexes (OCCs). If the number of OCCs was ʊ% of the expected number (as seen on TVS on the day of hCG trigger), then the oocytes were excluded from the study.
In vitro fertilization
We used single-step media as the IVF media in the study. The media used were that of SAGE, Cooper Surgical.
The different types of media that were used are as follows:
- Flushing media for retrieval, holding, and washing of oocytes
- Fertilization media (SAGE 1-Step with human albumin solution) for the IVF and culture of human gametes and embryos from fertilization until day 5/6 of development
- Sterile oil for tissue culture used in laboratory procedures that may require small volumes of tissue culture medium to be covered with a relatively inert, nonmiscible liquid component that allows diffusion of gases.
- Sperm preparation media kit
- Lower-phase gradient: 80% PureCeption™ with HEPES-buffered human tubal fluid (HTF-HEPES)
- Upper-phase gradient: 40% PureCeption™ with HTF-HEPES
- Sperm-washing medium: HTF-HEPES with human serum albumin, 5 mg/mL.
Protocol followed forin vitro fertilization
The following protocols were followed in the study.
Washing and segregation of oocytes
- The retrieved follicular fluid was emptied into an IVF grade 60-mm culture plate at 37°C. The OCCs were identified and segregated into a separate culture plate containing flushing media at 37°C
- The oocytes were repeatedly washed with flushing medium or preequilibrated culture medium, taking care to remove any blood clots and granulosa cells
- Then, they were transferred to preequilibrated culture medium (under oil) and stored at 37°C in a 6% CO2 environment.
Semen preparation was done by double-density gradient – swim-up technique.
- A prewash analysis was done, and the parameters of the semen sample were noted
- All components of the sperm preparation media kit and the semen sample were brought to 37°C before using. This avoided a cold “shock” to the spermatozoa
- The single-bilayered gradient was prepared by transferring 2.0 mL of lower phase (PureCeption™ 80%) into the conical tube. Then, 2.0 mL of upper phase (PureCeption™ 40%) was layered on top
- 2.5 mL of liquefied semen was gently placed onto the upper phase using a pipette or syringe
- The tube was then centrifuged for 20 min at 350 × g to 400 × g (or up to 750 × g for highly viscous samples)
- Following centrifugation, all layers except the lowest portion were removed (approximately 0.3 mL)
- 2–3 mL of sperm-washing medium was added, and the pellet was resuspended in a fresh centrifuge tube
- The tube was then centrifuged for 4–8 min at 250 × g. The higher number of sperms requires the maximum 8-min centrifugation time to ensure a complete and thorough sperm wash
- The supernatant was removed, and the pellet was then gently layered with 1–1.5 ml of 1-Step media and incubated at 37°C in an incubator in a 6% CO2 environment. This was done for a period of 8–10 min
- The top 0.8–1.2 ml of the supernatant media was removed and stored separately in a 5-ml (IVF grade) round-bottom tube with a loose cap and incubated at 37°C in an incubator in a 6% CO2 environment
- A small aliquot of the sample was tested for the postwash parameters of the semen sample.
Preparing plates for culture
Plates for cohort (group) culture
- The plates used were 60 mm (IVF grade) center well plate
- Oil and SAGE 1-Step media were incubated in loosely capped 14 ml (IVF grade) round bottom tube and incubated overnight at 37°C in an incubator in a 6% CO2 environment
- 1 ml (1000 μl) of SAGE 1-Step media was taken in the center well. This was overlaid with incubated oil.
Plates for microdroplet culture
- The plates used were 35 mm (IVF grade) plate
- Oil and SAGE 1-Step media were incubated in loosely capped 14 ml (IVF grade) round bottom tube and incubated overnight at 37°C in an incubator in a 6% CO2 environment
- The prepared semen sample was diluted with SAGE 1-Step media to get a sperm concentration of about 10–12 million/ml. This semen sample was used to prepare the microdroplets for the culture of oocytes.
- A central trough was prepared with SAGE 1-Step media running along one of the diameters of the plate. 25–30 μl droplets of the semen solution were laid circumferentially around the dish. This is then overlaid with incubated oil [Figure 1].
Insemination of oocytes
Insemination in cohort (group) culture plates
- 5–6 oocytes were placed in the prepared center well plates and incubated.
- After 20–30 min of incubation, the oocytes were inseminated with the prepared semen sample to achieve a concentration of about 1–1.5 million/ml/oocyte or about 10 million spermatozoa in the center well [Figure 2].
Insemination in microdroplet culture plates
- A single oocyte was placed in each of the microdroplet that had been prepared in the 35 mm plate.
Once the process of insemination was complete, the plates were incubated overnight at 37°C in an incubator in a 6% CO2 environment. The lids were loosely placed to prevent any accidental locking due to the oil overlay.
Culture of inseminated oocytes
The inseminated oocytes were then cultured further in a conventional incubator at 37°C in a 6% CO2 environment.
As per the protocols of conventional IVF, the oocytes were denuded of the cumulus covering around 18–20 h after insemination. Denudation was done using a denuding pipette of 170 μ. The time required to denude the inseminated oocytes in each arm was noted. The time taken was totaled, and the average time required to denude an inseminated oocyte was calculated.
The denuded oocytes of the group culture plates as well as microdroplet culture plates were washed separately in the trough containing the SAGE 1-Step media and then transferred into a similarly prepared plate and incubated overnight.
The number of pronuclei present in the oocytes was noted. This check was done after the denudation process was completed. Fertilization check was done at around 48 h after the insemination.
Assessment of fertilization
As per the criteria laid out for this study, the presence of at least a 2-cell embryo was considered as the sign of fertilization. All the plates were examined for the presence of embryos and their percentage noted.
Further culture of the oocytes
After the confirmation of fertilization at 48 h, the plates were further cultured for another 24 h, that is, a total of 72 h.
The resulting embryos were either transferred or frozen as per the clinical requirement of the patient.
Details of all cases were recorded on a structured format and analyzed with the help of registered version of SPSS IBM SPSS statistics, version 21. Group comparisons were made using Chi-square test (for categorical variables) or Student's t-test (for scalar variables). Statistical significance was assessed at P < 0.05.
| Results|| |
A total of 327 patients were included in the study, of which 159 patients were included in the cohort culture group. A total of 1032 oocytes were retrieved in this group. Of these, 621 had successful fertilization (as per the criteria selected). The time required for denudation on the day after insemination was also noted, and it totaled up to 28,973 s.
Of the 327 patients included in the study, 168 patients were in the microdrop culture group. A total of 1067 oocytes were retrieved in this group. Of these, 682 had successful fertilization (as per the criteria selected). The time required for denudation on the day after insemination was also noted, and it totalled up to 21,992 s [Table 1].
The fertilization rates were analyzed using the Chi-square test [Table 2]. In the cohort group, 621 of 1031 oocytes were fertilized, whereas 682 of 1067 were fertilized in the microdroplet group. The “P value” obtained showed that the difference in results obtained were not significant (P = 0.081988). It suggested that there was no difference in the fertilization rates between the two different methods of embryo culture.
The statistical significance of the time required for denudation was calculated using the Student's t-test for independent samples [Table 3]. The mean time for denudation of oocyte in the cohort and microdroplet groups was 27.86 and 20.61 s, respectively.
At P < 0.0001, the time taken to denude the oocytes of the microdroplet culture was found to be statistically significantly lesser than that of the cohort group. This is possibly due to the less time required to locate the oocyte in the microdroplet culture.
| Discussion|| |
Despite widespread technological advances, IVF remains a highly complex process with fertility centers internationally, all having the same common goal to achieve a live birth. While many aspects of IVF have been tested through evidence-based research, other facets have been less investigated with much variation in practice. One of the areas of focus in maximizing IVF success lies in the area of embryo culture techniques.
To supportin vitro development, different mammalian embryos have been empirically cultured in drops of medium with different volumes (e.g., 10–500 μl) covered with mineral oil. Recent studies showed that the volume of medium and embryo density (number per unit volume) are important factors that influence early embryo development.,,,, In IVF, different volumes of medium and numbers of embryos for incubation per drop have been described in literature with different results among different authors.
Extensive studies on mouse embryos by Lane andGardner and Salahuddin et al. have shown that group or cohort culture provides a better blastocyst formation rate.
Salahuddin et al. also suggested that the presence of unfertilized oocytes in the same culture medium droplet may hinder the further smooth development of the culturing embryos.
Many studies on human embryos,,, have carried out a comparison between cohort culture and microdroplet single culture. All the studies are based on the principle that there would be an increase of some embryo-derived growth or survival factor, which would influence the growth of the co-cultured embryos.
The above-mentioned studies,,, have mainly studied the culture from 8-cell to blastocyst stage. Rijnders et al. had suggested that there is no significant difference in outcome between the two culture systems during this period of culture. The other three studies have a beneficial trend toward the cohort group. However, this has a drawback that the individual embryo cannot be followed up during the process of incubation.
Despite extensive literature search, we could not find any article that addresses the influence of culture method on the process of fertilization.
The entire aim of this study was to find whether such an embryo-derived growth or survival factor was influencing the process of fertilization in the case of conventional IVF.
In our study, we did not find a statistically significant difference between the fertilization rates of cohort or microdrop culture techniques.
However, the time required to denude the inseminated oocyte was significantly lesser in the microdrop culture system. This would reduce the time that the plate is exposed to nonideal environment. This reduction of time will definitely have a beneficial effect on the subsequent outcome of the culture.
| Conclusions|| |
A comparative study of oocyte culture in cohort and microdroplet for fertilization rate in conventional IVF suggested that the difference in the fertilization rates of oocytes using these two different culture systems was not statistically significant. However, lesser time was required to denude the inseminated oocytes in case of microdroplet culture.
Even though it was not measured, the quantity of media used in microdroplet culture is lesser than that of cohort culture. The quantity of oil used in microdroplet culture is more than that of cohort culture.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]