Yimin Shu

Promotion of Human Early Embryonic Development and Blastocyst Outgrowth In Vitro Using Autocrine/Paracrine Growth Factors

Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6–8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner’s criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells.

The value of fast blastocoele re-expansion in the selection of a viable thawed blastocyst for transfer

OBJECTIVEnTo investigate the role of fast blastocoele re-expansion in the selection of viable thawed blastocysts for transfer.nnnDESIGNnRetrospective study.nnnSETTINGnAcademic assisted reproductive program.nnnPATIENT(S)nTransfer cycles were divided into two groups according to the presence or absence of fast re-expanded blastocysts. In group I (124 cycles), all transferred blastocysts had fast re-expanding blastocoele. In group II (113 cycles), no fast re-expanded blastocysts were included in the transfer.nnnINTERVENTION(S)nBlastocyst survival was defined as >50% of cells remaining intact after thaw and re-expansion after culture in vitro for 2-4 hours before transfer. Blastocysts with >or=50% re-expansion were designated as fast re-expanded blastocysts.nnnMAIN OUTCOME MEASURE(S)nPercentage of blastomere loss immediately after thaw, degree of blastocoele re-expansion, and clinical outcomes (pregnancy and implantation rates).nnnRESULT(S)nThe rates of survival and fast blastocoele re-expansion of partially intact blastocysts were significantly reduced as compared with fully intact blastocysts. Significantly higher rates of clinical pregnancy (37.1% vs. 16.8%) and implantation (26.7% vs. 11.3%) were obtained when all transferred blastocysts had fast re-expanding blastocoele as compared with those transfers without fast re-expanded blastocysts included.nnnCONCLUSION(S)nOur results showed that blastomere loss of thawed blastocyst was associated with a reduced ability to re-expand. As a discriminative morphologic marker of superior embryo viability, a fast re-expanded blastocyst would be given priority for transfer to better utilize the cryopreserved blastocysts.

Progesterone resistance in endometriosis: origins, consequences and interventions

Endometriosis is a common cause of pelvic pain and affects up to 10% of women of reproductive age. Aberrant progesterone signaling in the endometrium plays a significant role in impaired decidualization and establishment of ectopic endometrial implants. Eutopic endometrial cells from women with endometriosis fail to downregulate genes needed for decidualization, such as those involved in cell cycle regulation, leading to unbridled proliferation. Several causes of progesterone resistance in the endometrium have been postulated, including congenital “preconditioning”, whereby the in utero environment renders infants susceptible to neonatal uterine bleeding and endometriosis. Progesterone action is crucial to decreasing inflammation in the endometrium, and deviant progesterone signaling results in a proinflammatory phenotype. Conversely, chronic inflammation can induce a progesterone‐resistant state. Repetitive retrograde endometrial shedding begets chronic peritoneal inflammation, which further exacerbates progesterone resistance. Genetic causes of progesterone resistance include progesterone receptor gene polymorphisms, altered microRNA expression, and epigenetic modifications to progesterone receptors and their targets. Environmental toxins such as dioxin play a possible role in the genesis of endometriosis by permitting an inflammatory milieu. A consequence of impaired progesterone action is that hormonal therapy is rendered ineffective for a subset of women with endometriosis. Synthetic progestins, such as dienogest, may overcome this phenomenon by increasing progesterone receptor expression and decreasing proinflammatory cytokines. Other modalities include high dose depot formulations of progestins, medicated intrauterine devices and the likely advent of oral GnRH antagonists. Unearthing root causes of progesterone inaction in endometriosis will aid in the development of novel therapeutics geared toward prevention and treatment.

Expression and Potential Roles of HLA-G in Human Spermatogenesis and Early Embryonic Development

As one of the non-classical major histocompatibility complex(MHC)-1 antigens, Human Leukocyte Antigen G (HLA-G), has been suggested as a prognostic marker to identify the embryo developmental potential. In the present study, we investigated the potential roles of HLA-G in human spermatogenesis and early embryonic development. Quantitative real-time PCR analysis revealed that HLA-Gs expression was increased with increased Johnsen score in testicular tissues. There was no significant difference in HLA-G mRNA expression between testicular tissues with Johnsen score of 8–9 and normal sperm from ejaculated semen. HLA-G mRNA expression was detected in human zygotes, embryos and blastocysts but not in unfertilized oocytes. In testicular tissues where sperm was obtained by testicular sperm extraction (Johnsen score was 8 to 9), there were no correlations between HLA-G mRNA expression and fertilization, cleavage and high-quality embryo rates. At 48–72 h post-fertilization, HLA-G expression was higher in fast growing embryos. HLA-G specific siRNA injection into zygotes not only slowed down embryonic cleavage rate at 48 h post-fertilization, but also down-regulated the expression of embryo metabolism related gene (SLC2A1) and cell cycle-regulated gene (CCND2). Taken together, our findings suggested that HLA-G plays significant roles in human spermatogenesis and early embryonic development.

Cryopreservation of Human Oocytes and Embryos

With the advent of assisted reproductive technology, controled ovarian hyperstimulation (COH) is usually carried out to stimulate the growth of multiple follicles and produce multiple oocytes. Accordingly, multiple embryos are transferred to the uterus to increase the chances of success. However, multiple embryos can also increase the likelihood of multiple pregnancies, which are accompanied by a whole series of complications affecting both mother and child. There has been a trend toward transferring fewer embryos during the last decade. According to Center for Disease Control and Prevention (CDC), the average number of embryos transferred to women under 35 has dropped from 4.0 in 1995 to 2.8 in 2001 in the United States (N Engl J Med 350:1639-1645, 2004). In Europe, IVF centers in many countries have reduced the number of replaced embryos to two or even one (Ann N Y Acad Sci 1034:110-116, 2004). It is, therefore, very important to have a reliable technique to effectively cryopreserve the supernumerary embryos after transfer. The first pregnancy following the transfer of a frozen-thawed human embryo was reported by Trounson et al. in 1983 (Nature 305:707-709, 1983). Since then, embryo cryopreservation has become a very important part of the clinical use of in vitro fertilization. To date, human embryos have been successfully cryopreserved at the pronuclear, cleavage, and blastocyst stages of development. Moreover, recent advances in cryopreservation techniques have expanded the female fertility preservation from embryo to oocyte and ovarian tissue.

Pathogenesis of endometriosis: Interaction between Endocrine and inflammatory pathways

Despite an estimated prevalence of 11% in women and plausible historical descriptions dating back to the 17th century, the etiology of endometriosis remains poorly understood. Classical theories of the histological origins of endometriosis are reviewed below. Clinical presentations are variable, and signs and symptoms do not correlate well with the extent of disease. In this summary, we have attempted to synthesize the growing evidence that hormonal and immune factors conspire to activate a local inflammatory microenvironment that encourages endometriosis to persist and elaborate mediators of its two cardinal symptoms: pain and infertility. Surprisingly, in the search for novel therapeutics for medical treatment of endometriosis, some compounds appear to have dual pharmacological functions, simultaneously modifying the endocrine and immune system facets of this complex gynecologic syndrome. We predict that these lead drugs will provide more therapeutic choices for patients in the future.

Transfer of IVF-contaminated blastocysts with removal of the zona pellucida resulted in live births

PurposeBacterial contamination may cause loss or damage to cultured oocytes or embryos, resulting in cancelation or delaying of a fresh embryo transfer. While live births have been reported following the transfer of embryos contaminated with yeast, very little information is available on how to handle embryos with bacterial contamination. We report two cases of successful pregnancy in patients with bacterial contamination of embryo culture dishes.MethodsWe retrospectively reviewed 878 oocyte retrievals performed between January 2011 and December 2014. Bacterial contamination was recorded in two split IVF/ICSI cases, where contamination occurred in embryo culture drops containing embryos from conventional insemination but not from ICSI on day 3.ResultsTo minimize the adverse effects of bacterial contamination on transfer outcomes, we removed the zona pellucida of contaminated frozen blastocysts and successfully obtained clinical pregnancies following transfer of zona-free blastocysts that were previously contaminated during IVF culture.ConclusionsRemoval of the zona pellucida is an appropriate approach to handle blastocysts contaminated with bacteria during in vitro culture.

Stress and Implantation Failure

The cornerstone of female reproduction is the hypothalamic-pituitary-ovarian (HPO) axis. This term is incomplete in that it omits reference to the uterus and endometrium, which are clearly critical to the most important step in reproduction, namely, implantation. Implantation requires that the endometrium become receptive to a healthy blastocyst. Thus, folliculogenesis must result not only in ovulation but also in the secretion of estradiol and progesterone in a pattern that prepares the endometrium for implantation. While implantation failure can be defined as absent hCG production after ovulation that is signaled by menstrual bleeding, the definition also encompasses transient hCG production that is not sustained, which is often called “chemical” pregnancy.

Live birth of healthy twins following transfer of re-cryopreserved blastocysts developed from frozen embryos recovered from a malfunctioning freezer.

A computer-controlled cooling machine is the basic equipment required to perform slow freezing of human embryos. Although freezer malfunction is a rare event, it has been reported (HFEA press release. [1]). To date, there are no published reports regarding the fate of cryopreserved embryos encountered freezer malfunction during the slow cooling process. This case report describes our experience with a sudden temperature increase occurring during the slow cooling of human embryo cryopreservation. We successfully recovered cryopreserved embryos from the malfunctioning freezer by rapid thawing. Surviving embryos successfully developed to the blastocyst stage and were re-cryopreserved by vitrification. An ongoing twin pregnancy was achieved with two healthy twins following the transfer of two vitrified blastocysts.