This study was approved by the Ethical Committee of Guangdong Provincial People's
Hospital, Guangdong Academy of Medical Sciences. All methods were performed in accordance with the relevant guidelines and regulations and the approved protocol was followed throughout the study period.
For each couples entering the treatment cycle, we already signed an informed consent form to state the success rate and possible complications of IVF/ICSI, and informed them that their data might be used for scientific research but their personal information is completely confidential.
Differences between conventional 0PN and "0PN" in this paper
Conventionally, mature oocytes with no distinct two pronuclei (2PN) in the cytoplasm of any oocytes associated with extrusion of the second polar body at 16–20 h postinsemination are documented as 0PN. Accordingly, the time of the fertilization check is defined of 16–20 hour after insemination.
The observation point is delayed to more than 22 hours in L-R-ICSI because the time of reinsemination in L-R-ICSI is 5-6 hours earlier than normal insemination. Different from the traditional fertilization check, the observation point in this paper is more than 22 hours after insemination, therefore, "0PN" embryos in this article include but are not limited to 0PN embryos in the traditional sense, and we use quotation marks to distinguish them. That is to say, the "0PN" embryo in this article is compared to the traditional definition, which includes an embryo with prokaryotic disappearance between 20 hours after insemination and the observation point. This is also inconsistent with the standards of other articles on 0PN.
In L-R-ICSI of our center, 8 patients were successfully conceived and 9 babies were born, among which 5 were from 0PN, between 9 January 2015 and 23 August 2017. This paper mainly describes the data of 4 0PN cases, including a pair of twins.
Four female patients and their domestic partners with unexplained infertility for 3.4-7 years and aged from 26 to 32 years were scheduled for fertilization (Data of fresh cycles as table 1) . Three patients used the standard long stimulation GnRH agonist protocols and the other one used GnRH antagonist protocol. Human chorionic gonodotrophin at a dose of 5000 -10000 IU was administrated after at least two follicles of 18 mm or greater in size were visualized by means of transvaginal ultrasound scanning. Fifty cumulus oocyte complexes (COCs) were retrieved by ultrasound-guided needle aspiration in the four infertile couples. The COCs were collected and washed fourfold and transferred into a Center-Well Organ Culture Dish (353037,60×15 mm; Becton Dickinson, USA) containing 1 mL of G-IVF medium (Vitrolife, Sweden) for pre-insemination culture. Oocytes were inseminated in the same medium with approximately 3~4×105 motile spermatozoa/mL after hCG injection 40–42 hours later, and incubated at 37℃ in a mixed atmosphere of 6% CO2 and 5% O2 with high humidity. A fertilization check was performed 18-20 hours later (about 8 a.m.) on the next morning (D1) after insemination and only five oocytes were fertilized. After patients provided appropriate signed informed consent, L-R- ICSI as an alternative treatment was performed on the thirty-five metaphase II stage oocytes with sperm from D0 (day of oocyte pick-up, OPU) insemination at about 9.a.m.. Embryos were cultured in the G-1 medium drops (Vitrolife, Sweden), and covered with mineral oil in Falcon tissue culture dishes (353001, 35×10 mm, Becton Dickinson, USA). Around 8 a.m. on the D2 morning after OPU, only seven of these oocytes had two distinct pronuclei (2PN) in the cytoplasm, and the other 23 oocytes had no pronuclei but showed cleavage on the following culture.
All rescued embryos were observed on D3 or D5 or D6 after L-R- ICSI. Here, we introduced two systems of classifying the embryos. On day 3, the blastomeres and fragments were evaluated and classified as four types separately by means of the following scoring system. Blastomere: grade I-even sized blastomeres with regular morphology; grade II- slightly uneven sized blastomeres with regular morphology; grade III- asymmetrical blastomeres and irregular morphology; grade IV-severely asymmetrical blastomeres and irregular morphology or significant cytoplasmic particles. Embryo fragments were classified into I, II, III and IV grades according to fragmentation <5 %, 6%-20%, 21%-50% and >50 %. Number of cells 7-9, embryos and fragments were classified as I and II, but those with different levels of II were defined as top-quality embryos. Cell numbers >4, blastomeres grade I-III and fragments classified as I-II were considered suitable embryos for transfer, excluding embryos with blastomere as III and fragment as II. The blastocyst grading system proposed by Gardner and Schoolcraft was based on blastocyst morphology parameters ; that is, blastocyst development was divided into 6 stages according to the size of blastocoele expansion degree, blastocyst cavity and whether it hatched or not. The inner cell mass (ICM) and trophoblast ectoderm (TE) were classified into three levels: A, B and C according to cell number and intercellular adhesion (A: a good number of cells and good intercellular adhesion; B: small cell numbers and good intercellular adhesion; C: almost no cells). The blastocysts with blastocoeles were greater than or equal to 3, ICM and TE scores of A, B and C, but not both C, were considered as eligible and transferred, and those defined as good blastocysts for ICM and trophoblast ectoderm (TE) were graded A or B.
Avoiding dyssynchrony between the endometrium and embryonic development, fresh cycles of embryo transfers were cancelled. The couples were informed of the uncertainty associated with the transfer of 0PN-derived embryos in terms of the health of any resulting babies. All embryos and blastocysts that reached the standard of transfer, either from 2PN or 0PN, were frozen in vitrification. The freezing process between embryos and blastocysts differed in that the blastocysts needed to be shrunk artificially with a laser pulse prior to vitrification by the embryologist. Embryos/blastocysts were first transferred to an equilibration solution (VT101-①, KITAZATO, Japan) for 10 minutes, and subsequently transferred to a vitrification solution (VT101-②, KITAZATO, Japan) for 1 minute. Then, one or two embryos/blastocysts was/were placed into a cryotop (Cryotop ®, KITAZATO, Japan), which was quickly plunged into liquid nitrogen, and then covered with a tube and stored in liquid nitrogen.
About two months after oocyte pick-up, FET cycles were started, in which one or two cryopreserved embryos/blastocysts were warmed and transferred. To thaw, the embryos/blastocysts loaded into the cryotop were immersed in the first thawing solution (VT102-①, KITAZATO, Japan) at 37℃ for 1 minute, and then transferred to the second and the third thawing solutions (VT102-②, ③, KITAZATO, Japan) for 3 and 5 minutes at room temperature. Lastly, the embryos/blastocysts were washed in the fourth solution (VT102-④, KITAZATO, Japan) at 37 °C for 5 minutes. After warming, embryos/blastocysts with more than 50% intact blastomeres were regarded as viable and were transferred to the recipient uterus of all four patients.
Ten embryos/blastocysts of five FET cycles were transferred to the four patients. One patient received two transplants, the first of which had two 2-prokaryotic embryos, and was not a pregnancy. The remaining eight fresh cycles of 0 prokaryotic embryos were transplanted four times and four clinical pregnancies resulted (Data of of FET cycles as table 2). Then, 6-8 weeks after FET, four clinical pregnancies were confirmed using transvaginal ultrasonography. Among them were two twin pregnancies with four sacs but three fetal heartbeats.