Y. Kuang

Luteal-phase ovarian stimulation is feasible for producing competent oocytes in women undergoing in vitro fertilization/intracytoplasmic sperm injection treatment, with optimal pregnancy outcomes in frozen-thawed embryo transfer cycles.

OBJECTIVEnTo explore the feasibility of luteal-phase ovarian stimulation using hMG and letrozole in terms of ovarian response and pregnancy outcome using frozen-thawed embryo transfer.nnnDESIGNnA prospective cohort study.nnnSETTINGnAcademic tertiary-care medical center.nnnPATIENT(S)nTwo hundred forty-two female patients undergoing IVF/intracytoplasmic sperm injection (ICSI) treatment.nnnINTERVENTION(S)nOvarian stimulation was initiated with hMG 225 IU and letrozole 2.5 mg daily after spontaneous ovulation. Letrozole administration was stopped when the dominant follicles reached diameters of 12 mm. Ovulation was induced with a GnRH agonist 100 μg when at least three follicles reached diameters of 18 mm or one dominant follicle reached 20 mm. The highest quality embryos were extracted and cryopreserved for later transfer.nnnMAIN OUTCOME MEASURE(S)nThe primary outcome measured was the number of oocytes retrieved. Secondary outcomes were the clinical pregnancy rate, ongoing pregnancy rate, and implantation rate after frozen embryo transfer (FET) cycles.nnnRESULT(S)nOf the 242 women enrolled in the study, all participants succeeded in producing oocytes and 227 women had highest-quality embryos to cryopreserve. The average number of oocytes retrieved was 13.1, producing an average of 4.8 highest quality embryos. Moreover, no cases experienced a premature LH surge or moderate/severe ovarian hyperstimulation syndrome during the stimulation cycles. In FETs, the clinical pregnancy rate, ongoing pregnancy rate, and implantation rate were 55.46% (127/229), 48.91% (112/229), and 40.37% (174/431), respectively. Of all the pregnancies in the study, 68 resulted in live births and 44 were ongoing.nnnCONCLUSION(S)nLuteal-phase ovarian stimulation is feasible for producing competent oocytes/embryos in women undergoing IVF/ICSI treatments, with optimal pregnancy outcomes in FET cycles.

Double stimulations during the follicular and luteal phases of poor responders in IVF/ICSI programmes (Shanghai protocol)

Previous studies have shown that existing antral follicles in the luteal phase enable ovarian stimulation. In a pilot study, the efficacy of double stimulations during the follicular and luteal phases in women with poor ovarian response was explored (defined according to the Bologna criteria). Thirty-eight women began with mild ovarian stimulation. After the first oocyte retrieval, human menopausal gonadotrophin and letrozole were administrated to stimulate follicle development, and oocyte retrieval was carried out a second time when dominant follicles had matured. The primary outcome measured was the number of oocytes retrieved: stage one 1.7 ± 1.0; stage two 3.5 ± 3.2. From the double stimulation, 167 oocytes were collected and 26 out of 38 (68.4%) succeeded in producing one to six viable embryos cryopreserved for later transfer. Twenty-one women underwent 23 cryopreserved embryo transfers, resulting in 13 clinical pregnancies. The study shows that double ovarian stimulations in the same menstrual cycle provide more opportunities for retrieving oocytes in poor responders. The stimulation can start in the luteal phase resulting in retrieval of more oocytes in a short period of time. This offers new hope for women with poor ovarian response and newly diagnosed cancer patients needing fertility preservation.

Secretome profile of mouse oocytes after activation using mass spectrum

ObjectiveMammalian oocytes undergo a cortical reaction after fertilization, releasing cortical granules and other proteins into the perivitelline space and inhibiting polyspermy. Few studies have evaluated the biological functions and properties of these proteins.Study designWe investigated mouse oocytes in which the zona pellucida (ZP) was present (ZP-intact group) or absent (ZP-free group).ResultsAfter being activated by Srcl2, secreted proteins are collected from mouse oocytes. Mass spectrometry analysis was performed that identified proteins such as Ldhb, PADi6, Uchl1, Pebp1, Alb, Hsp90aa1, Prss1, trypsinogen 7, trypsin 4, trypsin 10, Sod1, Zp1, Zp2, Zp3, Akap8, Npm2, Pkm2 and Ppia in the ZP-free group. Proteins such as Ldhb, Uchl1, Prss1, trypsin 10, trypsinogen 7, and Ast1 were identified in the ZP-intact groups. The expression of some proteins, including Ldhb, Alb and Sod1, were initially detected following oocyte activation. The finding of four trypsin subtypes, such as Prss1, further support previous observations. Studies investigating the physiological functions and properties of these proteins are ongoing.ConclusionsResearch on these cortical proteins provides a theoretical basis for understanding polyspermy inhibition at the level of ZP and gives technological support for fertilization detection, assessment of oocyte quality and embryonic culture.

New technique for mouse oocyte injection via a modified holding pipette

To improve mouse oocyte survival from intracytoplasmic sperm injection, the sharp tip of the injection pipette has been modified to have a flat end. Here, for the same goal but for a more convenient manipulation, a sharp injection pipette was kept whereas the holding pipette was modified to have a trumpet-shaped opening, which allows deeper injection into the oocyte as it is held. Mouse oocyte injection with mouse and human spermatozoa was performed at 37°C. For the injection of mouse oocyte with mouse sperm head, a significantly higher survival rate (83%) was achieved by utilizing the modified holding pipette than the conventional one (21%; P<0.001) and the fertilization rates were normal and comparable for both methods (82% versus 81%). A superior survival rate (82%) and acceptable normal fertilization rate (71%) were also achieved by utilizing the modified holding pipette for interspecies ICSI (injecting mouse oocyte with human spermatozoon). Taken together, by utilizing a holding pipette with a trumpet-shaped opening, acceptable rates of mouse oocyte survival and fertilization can be achieved using a sharp injection pipette under conditions usual for human oocyte injection.

A novel method for cryopreservation of individual human spermatozoa

The purpose of this study is to develop a novel method for the cryopreservation and efficient post-thaw recovery of individual or small numbers of human spermatozoa. Spermatozoa equilibrated in cryoprotectant buffer were injected with an intracytoplasmic sperm injection (ICSI) needle into a droplet of cryoprotectant on a homemade cryoleaf. The droplet was of cryoprotectant and seminal plasma at a ratio of 1:1. The sperm-loaded cryoleaf was slowly lowered over and stored in liquid nitrogen. Spermatozoa were thawed in a 37°C oil bath without dilution and centrifugation. To test the fertilizing ability of these spermatozoa, the recovered spermatozoa were injected by ICSI into 1-d-old or in vitro-matured human oocytes. Fresh spermatozoa from the same semen samples served as controls. The trials were performed in two separate experiments. In the first set of experiments, 92 spermatozoa were thawed and carefully investigated. The spermatozoa from percutaneous epididymal sperm aspiration had a motility recovery of 92.9% (13/14); ejaculated spermatozoa had a motility recovery of 61.5% (48/78), and only 1.3% (1/78) was lost. Together in the first and second set of experiments, the fertilization rates for the fresh and frozen–thawed spermatozoa were 67.6% (25/37) and 60.6% (40/66), respectively (Pu2009=u20090.052). The mean embryo cleavage rates in the fresh and frozen–thawed groups were 88% (22/25) and 85% (34/40), respectively (Pu2009=u20090.990). This cryopreservation method for individual or small numbers of human spermatozoa was efficient and simple. These findings make this method a promising technique for the clinical application of ejaculated sperm from oligozoospermic patients.

Enhancement of the efficiency of oocyte vitrification through regulation of histone deacetylase 6 expression

ObjectiveSuccessful oocyte vitrification (OV) is critical for cryopreservation of the oocytes from female patients with infertility, polycystic ovaries, and gynecologic cancers. Recent evidence suggests that relatively low levels of histone acetylation are critical for maintenance of the maturation capacity of cryopreserved oocytes. However, previous studies have only demonstrated a key role of histone deacetylases (HDAC) 1 and 2 in the cryopreservation of oocytes.MethodsIn this study, we investigated the role of HDAC6 in these settings. We found that mouse oocytes with low HDAC6 levels decreased survival rate, cleavage rate, and blastocyst rate after OV. Bioinformatics analyses were used to predict HDAC6-targeting microRNAs (miRNAs), while the functional binding of miRNAs to HDAC6 mRNA was evaluated by a dual luciferase reporter assay.ResultsAmong all HDAC6-targeting miRNAs, we detected expression of miR-558, miR-527, and miR-762 in mouse oocytes. Specifically, we found that only miR-762 significantly inhibited protein translation of HDAC6 via binding to the 3′-UTR of the HDAC6 mRNA. Transfection of oocytes with HDAC6 or antisense of miR-762 significantly increased the survival rate, the cleavage rate, and blastocyst rate after OV.ConclusionAs a result, our data suggest that induction of HDAC6 levels by miR-762 suppression may improve the current protocol for OV.