This study investigated whether adding outer-well medium to inhibit osmotic changes in culture media in a dry-type incubator improved the clinical outcomes of
In culture dishes, the osmotic changes in media (20 µL)-covered oil with or without outer-well medium (humid or dry culture conditions, respectively) were compared after 3 days of incubation in a dry-type incubator. One-step (Origio) and G1/G2 (Vitrolife) media were used.
The osmotic changes in the dry culture condition (308 mOsm) were higher than in the humid culture conditions (285–290 mOsm) after 3 days of incubation. In day 3 IVF-ET cycles, although the pregnancy rate did not significantly differ between the dry (46.2%) and humid culture (51.0%) groups, the rates of abortion and ongoing pregnancy were significantly better in the humid culture group (1.5% and 49.5%, respectively) than in the dry culture group (8.3% and 37.8%, respectively,
Hyperosmotic changes in media occurred in a dry-type incubator by evaporation, although the medium was covered with oil. These osmotic changes were efficiently inhibited by supplementation of outer-well medium, which resulted in improved pregnancy outcomes.
Even brief exposure of preimplantation mouse embryos to high-osmolality culture medium (>300 mOsm/kg) in the absence of osmolytes resulted in impaired development [
From a different point of view, an increase in extracellular osmolality can promote water flux out of the cell, triggering cell shrinkage and intracellular dehydration [
The present study was performed to compare osmotic changes in culture media covered in oil in various types of culture dishes, and to investigate whether a beneficial effect on clinical outcomes in
This retrospective study was approved by the Institutional Review Board of Mamapapa and Baby Clinic (IRB No. 2019-10-01), and was conducted from August 2018 to August 2019.
In total, 796 IVF-ET cycles in 673 patients were analyzed in the present study. Twelve patients who underwent their first 3-day IVF-ET cycles using a cell culture dish, but failed to show implantation or ongoing pregnancy, completed their second IVF-ET cycles using a GPS dish to compare the clinical outcomes between cell culture and GPS dish cycles. After a comparison of these 12 patients, we changed the culture medium from 1-Step medium to G1/G2 medium; the remaining 772 IVF-ET cycles in 661 patients were performed using G1/G2 medium. Of these cycles, 168 IVF-ET cycles (156 day 3 IVF-ET cycles + 12 day 5 IVF-ET cycles) in 159 patients used cell culture dishes, while 628 IVF-ET cycles (539 day 3 IVF-ET cycles + 89 day 5 IVF-ET cycles) in 514 patients used GPS dishes. In the early stage of the study, we found that using the GPS dish had a beneficial effect on clinical outcomes, and after confirming a significant improvement in pregnancy outcomes in the GPS dish group, we completely changed to GPS dishes. The ultimate goal of our studies is to improve the pregnancy rate in IVF cycles; therefore, we could no longer use the cell culture dishes. This change resulted in the difference of the number of cycles between the cell culture and GPS dish groups.
Controlled stimulation for IVF cycles was performed with a mild stimulation protocol using a combination of a gonadotropin-releasing hormone (GnRH) antagonist and gonadotropins. Patients received 150 IU of recombinant follicle-stimulating hormone (Gonal-F; Merck Serono, Darmstadt, Germany) alone as a daily injection from cycle day 3 until the day when human chorionic gonadotropin (hCG) was administered. The GnRH antagonist (Cetrotide, Merck Serono) was initiated on the day when the leading follicle reached a diameter of 14 mm. Ovarian follicular development was monitored by transvaginal ultrasonography. When the leading follicles reached ≥18 mm in maximum diameter, as detected by sonography, ovulation was induced by injecting 250 µg of hCG (Ovidrel, Merck Serono). Oocyte retrieval was performed using 20-gauge ovum aspiration needles (Cook Medical, Bloomington, IN, USA) under standard transvaginal ultrasound guidance 35–36 hours after hCG administration. The luteal phase was supported by progesterone injection or vaginal gel (Crinone, Merck Serono). A serum β-hCG test was performed about 2 weeks after oocyte retrieval. Clinical pregnancy was confirmed by the visualization of a gestational sac. Ongoing pregnancy was defined as a pregnancy that was maintained for over 20 weeks of gestation.
1-Step (Origio, Malov, Denmark) and G1/G2 media (Vitrolife, Göteborg, Sweden) were employed in IVF-ET cycles. The culture dishes for IVF and embryo culture were prepared and incubated in a humid-type incubator (HERAcell 150i; Thermo Scientific, Waltham, MA, USA) overnight to achieve an optimal pH of 7.2–7.3. Fertilized oocytes were individually cultured in 20-μL drops of the culture medium covered in oil for 3–5 days until transfer, in 6.0% CO2, 5% O2 and 89.0% N2, in a dry-type incubator (Miri; ESCO, New Haven, CT, USA). The embryos were cultured in a cell culture dish without outer-well medium (dry culture condition) or in a µ-drop GPS dish with outer-well medium (humid culture condition). In day 5 IVF-ET cycles of the cell culture dish and GPS dish groups, dish change was performed on day 3 by transferring the embryos to a new culture dish prepared on day 2, to inhibit osmotic changes induced by evaporation and to serve as a new culture medium.
Osmotic changes in micro-drops (20 µL) of medium covered in oil and with or without outer-well medium supplementation were compared in the following types of culture dishes: cell culture (Corning Inc., Corning, NY, USA), µ-droplet culture (Vitrolife), microwell culture (DNP, Kashiwa, Japan), and µ-drop GPS (LifeGlobal, Brussels, Belgium) (
The quality of embryos was daily evaluated and scored according to the developmental stage and speed, as well as the shape of blastomeres and degree of fragmentation (
Statistical analysis was performed with SPSS ver. 11.0 (SPSS Inc., Chicago, IL, USA). Means and standard deviations were calculated for all variables. The Student
Osmotic changes in 20-µL droplets of Ham’s F-10 (280 mOsm) and G1 (275 mOsm) media according to the various types of culture dishes were compared after 3 days of incubation in the dry-type incubator (
As shown in
There were no significant differences in the characteristics of the same 12 patients between the first and second IVF-ET cycles, because the second IVF-ET cycles were performed within 6 months after the completion of the first IVF cycles. Although the rates of clinical pregnancy and abortion (50.0% and 0%) in the second IVF-ET cycles were more favorable than those (30.7% and 30.7%, respectively) in the first IVF-ET cycles, the difference was not statistically significant. However, the ongoing pregnancy rate (50.0%) of the second IVF-ET cycles was significantly higher than the rate (0%,
In the day 3 IVF-ET cycles, there were no significant differences in the mean age (36.9±4.4 and 37.3±4.2), endometrial thickness (10.6±2.4 and 10.4±2.0 mm), score of transferred embryos (9.1±2.3 and 8.9±2.4) and fertilization rate (67.4 and 69.4%) between the dry (156 cycles) and humid culture (539 cycles) groups (
In the day 5 IVF-ET cycles, there were also no differences in the mean age (36.1±3.9 and 36.3±3.4 years, respectively), endometrial thickness (10.8±1.7 and 10.6±2.1 mm, respectively), the number (1.0±0.0 and 1.0±0.2, respectively) and score (16.5±1.8 and 15.3±3.0, respectively) of embryos transferred, or the fertilization rate (77.7% and 76.2%, respectively) between the dry (12 cycles) and humid culture (89 cycles) groups (
When the osmolality of the medium increases above a certain threshold, embryo development is compromised [
One possible mechanism is the use of various amino acids as organic osmolytes by embryos. The addition of various organic osmolytes, including taurine [
Baltz and Tartia [
In the present study, after 3 days of incubation, even under an oil layer, the osmolality of media in the dry culture condition (301.1 mOsm) was higher than that of media in the humid culture condition (285.3–290.4 mOsm) in the dry-type incubator. Gasperin et al. [
In the present study, in day 5 ET cycles, the rates of clinical and ongoing pregnancy in the humid culture group were higher than the rates in the dry culture group. Similarly, no meaningful difference was found in the clinical and ongoing pregnancy rates of the humid culture groups in day 5 ET cycles (57% and 52%) between the previous [
Fawzy et al. [
There was no significant difference in the development of early-stage mouse embryos cultured between the high-osmolality (310–330 mOsm) and low-osmolality (270–290 mOsm) media. However, in the development of late-stage embryos, the embryos cultured in the low-osmolality medium showed a significantly higher blastocyst formation rate than those cultured in the high-osmolality medium [
When a cell shrinks in hyperosmotic conditions, inorganic ions (Na+, K+) are accumulated via osmotically regulated ion transporters [
We used G1/G2 medium (Vitrolife), which contains 135–145 µM glycine [
In conclusion, the dry culture condition showed a higher osmotic change in the medium than the humid culture condition, even though the media were covered in oil. Hyperosmotic changes showed a detrimental effect on clinical outcomes in human IVF-ET cycles. This hyperosmotic stress could be alleviated by supplementation with outer-well medium to maintain the optimal osmolality of medium, which resulted in the improvement of clinical outcomes in IVF-ET cycles. We are preparing a follow-up study to investigate relationships between the osmolality of culture media and specific patterns of gene expression in porcine embryos.
No potential conflict of interest relevant to this article was reported.
Conceptualization: **. Data curation: **. Formal analysis: **. Funding acquisition: **. Methodology: **. Project administration: **. Visualization: **. Writing–original draft: **. Writing–review & editing: **.
Various culture dishes were used to compare osmotic changes of μ-drop medium covered in oil and with (humid culture) or without outer well medium (dry culture) after 3 days of incubation in a dry-type incubator.
Embryo sequential scoring for evaluation of embryo quality. ICM, inner cell mass.
Osmotic change of media according to the type of culture dishes after 3 days incubation in dry type incubator
Culture dish | Incubator type | Media (mOsm) | Cover oil (mL) | Outer well media (mL) | Osmolality (mOsm/kg) |
---|---|---|---|---|---|
Cell culture (Corning) | Humid | Ham’s F-10 (280) | 6 | 0 | 286.1 |
Cell culture (Corning) | Dry | Ham’s F-10 (280) | 6 | 0 | 301.1 |
µ-droplet culture (Vitrolife) | Dry | Ham’s F-10 (280) | 0.8 | 0.9 | 290.4 |
Microwell culture (DNP) | Dry | Ham’s F-10 (280) | 0.03 | 2 | 285.3 |
µ-drop GPS (LifeGlobal) | Dry | Ham’s F-10 (280) | 2 | 4 | 287.2 |
Cell culture (Corning) | Dry | G1 (275) | 6 | 0 | 293.7 |
µ-drop GPS (LifeGlobal) | Dry | G1 (275) | 2 | 0 | 293.6 |
µ-drop GPS (LifeGlobal) | Dry | G1 (275) | 2 | 4 | 285.0 |
Humid, HERAcell 150i incubator; Dry, ESCO Miri R incubator.
Clinical outcomes of the 1st (cell culture dish, Dry culture) and 2nd Day 3 IVF-ET cycles (GPS dish, Humid culture) in the same 12 patients using 1-step (Origio) medium
1-step medium/day 3 ET (dry-type incubator) | First IVF cycle (cell culture dish) |
Second IVF cycle (GPS dish) |
|||||
---|---|---|---|---|---|---|---|
ICSI | cIVF | Total | ICSI | cIVF | Total | ||
No. of IVF cycles | 8 | 4 | 12 | 9 | 3 | 12 | |
Mean age (yr) | 40.1 | 35 | 38.4±4.9 | 40.3 | 33.3 | 38.5±4.9 | 0.467 |
Mean endometrium thickness (mm) | 9.9 | 9.3 | 9.7±1.7 | 9.8 | 11.1 | 10.1±2.1 | 0.300 |
Mean no. of oocytes aspirated | 4.5 | 7.7 | 5.5±3.9 | 5.4 | 6 | 5.5±2.6 | 0.500 |
2PN oocyte | 26/36 (72.2) | 17/31 (54.8) | 43/67 (71.6) | 29/49 (59.1) | 14/18 (77.7) | 43/67 (74.1) | 0.924 |
Mean no. of embryos transferred | 1.7 | 2.0 | 1.8±0.3 | 2.0 | 2.0 | 2.0±0.0 | 0.076 |
Mean score of embryos transferred | 7.3 | 8.3 | 7.6±2.2 | 6.4 | 8.0 | 6.8±2.7 | 0.211 |
Clinical pregnancy cycle | 3 (37.5) | 1 (25.0) | 4 (30.7) | 4 (44.4) | 2 (66.6) | 6 (50.0) | 0.678 |
Abortion cycle | 3 (37.5) | 1 (25.0) | 4 (30.7) | 0 | 0 | 0 | 0.100 |
Ongoing pregnancy | 0 | 0 | 0 | 4 (44.4) | 2 (66.6) | 6 (50.0) |
0.018 |
Values are presented as mean±standard deviation or number (%).
Dry culture, without outer well medium; Humid culture, with outer well medium; IVF, in vitro fertilization; ET, embryo transfer; ICSI, intracytoplasmic sperm injection; cIVF, conventional IVF; 2PN, two pronuclei.
Clinical outcomes of day 3 IVF-ET cycles in Cell culture dish (Dry culture) and GPS dish (Humid culture) groups using G1/G2 media (Vitrolife)
G1/G2 media/day 3 ET (dry-type incubator) | Cell culture dish (dry culture) |
GPS dish (humid culture) |
|||||
---|---|---|---|---|---|---|---|
ICSI | cIVF | Total | ICSI | cIVF | Total | ||
No. of IVF cycles | 82 | 74 | 156 | 236 | 303 | 539 | |
Mean age (yr) | 37.3 | 36.6 | 36.9±4.4 | 38.2 | 36.5 | 37.3±4.2 | 0.434 |
Mean endometrium thickness (mm) | 10.6 | 10.6 | 10.6±2.4 | 14.2 | 10.5 | 10.4±2.0 | 0.354 |
Mean no. of oocytes aspirated | 7.9 |
8.9a) | 8.4±4.4 |
6.4 | 8.0 | 7.3±4.0 | 0.003 |
2PN oocyte | 411 (63.1) | 471 (71.5) | 882 (67.4) | 992 (65.7) | 1,737 (71.7) | 2,729 (69.4) | 0.177 |
Mean no. of embryos transferred | 1.9a) | 2.0 | 2.0±0.4 |
1.8 | 2.0 | 1.9±0.3 | 0.013 |
Mean score of embryos transferred | 8.8 | 9.4 | 9.1±2.3 | 8.4 | 9.2 | 8.9±2.4 | 0.273 |
Clinical pregnancy cycles | 36 (43.9) | 36 (48.6) | 72 (46.2) | 103 (43.6) | 172 (56.7) | 275 (51.0) | 0.337 |
Abortion cycle | 7 (8.5) |
6 (8.1) |
13 (8.3) |
3 (1.3) | 5 (1.7) | 8 (1.5) | <0.001 |
Ongoing pregnancy | 29 (35.3) | 30 (40.5) | 59 (37.8) | 100 (42.3) | 167 (55.1) |
267 (49.5) |
0.012 |
Values are presented as mean±standard deviation or number (%).
IVF-ET,
Clinical outcomes of day 5 IVF-ET cycles in Cell culture dish (Dry culture) and GPS dish (Humid culture) groups using G1/G2 media
G1/G2 media/day 5 ET (dry-type incubator) | Cell culture dish (dry culture) | GPS dish (humid culture) | |||||
---|---|---|---|---|---|---|---|
ICSI | cIVF | Total | ICSI | cIVF | Total | ||
No. of IVF cycles | 5 | 7 | 12 | 28 | 61 | 89 | |
Mean age (yr) | 36.6 | 35.7 | 36.1±3.9 | 36.8 | 36 | 36.3±3.4 | 0.846 |
Mean endometrium thickness (mm) | 9.84 | 11.5 | 10.8±1.7 | 10.8 | 10.5 | 10.6±2.1 | 0.701 |
Mean no. of oocytes aspirated | 12.8 |
12 | 12.3±4.7 | 9 | 10.2 | 9.9±3.4 | 0.026 |
2PN oocyte (%) | 51 (79.6) | 64 (76.1) | 115 (77.7) | 190 (75.3) | 480 (76.5) | 670 (76.2) | 0.773 |
Mean no. of embryos transferred | 1.0 | 1.0 | 1.0±0.0 | 1.0 | 1.0 | 1.0±0.2 | 0.459 |
Mean score of embryos transferred | 16.4 | 16.6 | 16.5±1.8 | 14.5 | 15.6 | 15.3±3.0 | 0.168 |
Clinical pregnancy cycles | 3 (60.0) | 3 (42.8) | 6 (50.0) | 14 (50.0) | 39 (63.9) | 53 (59.5) | 0.750 |
Abortion cycle | 1 (20.0) | 2 (28.5) | 3 (25.0) |
0 | 2 (3.3) | 2 (2.2) | 0.006 |
Ongoing pregnancy | 2 (40.0) | 1 (14.2) | 3 (25.0) | 14 (50.0) | 37 (60.6) | 51 (57.3) | 0.072 |
Values are presented as mean±standard deviation or number (%).
IVF-ET,