| Controlled ovarian hyperstimulation (COH) was the most important part in assisted reproduction techniques (ART). Gonadotrophin-releasing hormone agonists (GnRHa) have been the gold standard in stimulation protocols for many years. Gonadotrophin-releasing hormone antagonists (GnRH-A) can be used to prevent a luteinizing hormone (LH) surge during COH without the hypo-estrogenic side-effects, flare-up, or long down-regulation period associated with GnRH agonists. The updated Cochrane database in2011suggests that GnRH-A protocol is associated with a significant reduction in OHSS and comparable clinical outcomes. The future paramount aim should be to further improve the reproductive outcome by optimizing the details of the GnRH-A protocol. LH supplementation in GnRH-A protocol was studied widely in improving the impaired follicle development due to low LH level induced by GnRH-A.Human chorionic gonadotropin (hCG) was called the wonder of today’s science. Firstly, because hCG is such an extreme molecule. hCG is the most acidic glycoprotein containing the highest proportion of sugars. Secondly, hCG exists in5common forms. Finally, it has so many functions ranging from control of human pregnancy to human cancer.HCG has been used as a substitute for the luteinizing hormone (LH) surge because of the degree of homology between the two hormones. Recently, the LH/hCG receptor has been found an almost ubiquitous distribution in reproductive organs, thus suggesting that the actions of hCG might be more extensive than previously thought. In addition, hCG has a slower plasma metabolic clearance, and may be more effective than LH. The longer half-life and greater affinity for the LH/hCG receptor of hCG account for a potency ratio estimate of hCG-to-LH of around1：6. The application of hCG in GnRH-A protocol need further investigation.It has been reported that low-dose hCG can support development and maturation of larger ovarian follicles independently of follicular stimulation hormone (FSH), and the pre-treatment of low-dose hCG before or in the early stage of controlled ovarian hyperstimulation (COH) might provide an effective way in reducing rFSH use and enhancing oocyte developmental competence to obtain top-quality embryos, and improves implantation and on-going pregnancy rates.Several studies have shown that hCG can replace rFSH during the final days of controlled ovarian stimulation (COS)(Filicori et al.,2005; Blockeel et al.,2009; Kosmas et al.,2009), but seldom study has investigated the effects of addition of hCG to rFSH from the first day of stimulation.Prospective multicentre studies and meta-analyses have shown that pregnancy and live birth rates may be improved after IVF by using highly purified-human menopausal gonadotrophins (HP-hMG) rather than r-FSH (Andersen et al.,2006; Platteau et al.,2008; van Wely et al.,2011). HP-hMG contains hCG and compared with rFSH, COS with HP-hMG has been shown to induce a different endocrine profile, different follicular dynamics, a larger proportion of top-quality embryos and more favourable endometrial receptivity (Andersen et al.,2006; Smitz et al.,2007; Ziebe et al.,2007).The LH activity of urinary-derived commercially available menotrophins varies, but in the HP menotrophins (hMG; Menopur), the main LH activity is related to hCG and for75IU of FSH, the drug contains around10IU of hCG (Wolfenson et al.,2005). When using HP-hMG for COS, hCG will be present throughout the stimulation. The differences in endocrine profiles observed using HP-hMG versus rFSH have in part been attributed to the hCG content. The circulating levels of hCG on Day6in the long agonist protocol have been shown to be positively correlated with live birth rates and number of top-quality embryos (Smitz et al.,2007).HCG was the first reported bio-marker embryonic secretion, which is primarily produced by the embryo and later by the syncytiotrophoblast. The hormone was detected at various levels in embryonic culture media from day2to blastocyst stage by different assays. The ability to dectect hCG from day2spent culture media may be used as a marker for embryo competence. However, they were only used in research now, and no use to compare the different COH protocols. Recently, the LH/hCG receptor has been found an almost ubiquitous distribution in reproductive organs. The study demonstrates the mRNA expression of the LHR in human oocytes and preimplantation embryo. But gene expression does not necessarily imply that the transcripts are translated in protein or the receptors are functionally invoved in signal transduction.The purpose of the present study was to determine whether low dose hCG early added to rFSH in regimens of ovarian stimulation could produce better results compare to rFSH alone in IVF-ET antagonist protocol.This was investigated through a regular study analysing the clinical, embryological and endocrine aspects. An additional aim was to define possible ceiling levels, i.e. levels above which there were no additional beneficial effects or potentially harmful effects of supplementation with hCG. The primary end-point was the number of top-quality embryos per patient on Day3after fertilization. The second objective of our study was to develop an in-house enzyme-linked immunoscorbent assay (ELASA) procedure to detect and qualify hCG from day3spent embryo culture media and to determine the association of hCG with the development potencial of the embryo and compare the outcome of different COH protocols. The third objective of the study was to develop a blastocyst biopsy combined immunohistochemical technique procedure to detect the protein expression of LH/HCG receptor in human preimplation embryo. To evaluate the effluence on embryo development by the number of top-quality embryos, the value of hCG from day3spent embryo culture media and the protein expression of LH/HCG receptor in day3discarded embryos in early addition of low dose human chorionic gonadotropin during controlled ovarian hyperstimulation antagonist protocol.Materials and methodsStudy design:This prospective, single center, paired design controlled trial was performed on100patients undergoing IVF treatment in Reproductive Center,Guangdong General Hospital,from March2012to November2012. The study was approved by the institutional review board and Ethics Committee. All the participants were informed and signed the consent.Patients and treatment protocol100patients undergoing COH in a GnRH-antagonist protocol was paired designed into two group according to age, primary infertility, infertility duration, the number of antral follical count and infertility factor.The Control group received a standard treatment with rFSH (Purgen) plus a GnRH-antagonist, daily from Day6of stimulation. In the study group, low dose HCG100IU was added to rFSH in the first day of stimulation.Both groups were started at a dose of150-600IU per day according the AFC on the2day of period. Transvaginal ultrasound was performed after4days of stimulation, and the dose of recombinant FSH and hCG was adjusted based on the number and size of follicles and the estradiol level. A daily morning dose of250mg of ganirelix acetate was started after Day6of stimulation. When there were at least two follicles with a mean diameter of18mm, with at least two additional follicles sized16mm, hCG was administered (Ovidel250ug). Oocyte retrieval was performed36hours later, and the embryos were transferred either3days after retrieval, depending on embryo number and quality. Luteal phase support was maintained with600mg of progesterone intravaginal daily beginning the evening after the oocyte retrieval and continuing until7to8weeks’ estimated gestational age.Serum LH, progestone and estradiol levels were measured at several intervals after the start of GnRH antagonist, and on the day of hCG administration for final oocyte maturation. All serum tests were drawn in the morning before the morning doses of gonadotropins or ganirelix acetate were administered. Luteinizing hormone and estradiol were measured by using an electrochemiluminescence immunoassay (Modular Analytics E170module; Roche).Data collection:1. Clinical outcome:duration of stimulation, total dose of rFSH,2. Lab outcome:follicular development, number of oocytes retrieved, number of M2oocytes, fertilization, fertilization rate, number of embryos transferred, implantation rate, number of top-quality embryos on Day3.3. Serum endocrinology:basal FSH and LH, stimulate day LH, P, E2, HCG value; HCG day LH, E2, P and HCG value.4. Pregnancy:pregnancy rates and miscarriage rates5. Side effect and OHSS case.Study end-pointsThe primary end-point was the total number of top-quality embryos on Day3. A top-quality embryo was defined as four to five blastomeres on Day2, seven or more blastomeres on Day3, equally sized blastomeres and≤20%fragmentation on Day3and no multinucleation. These endpoints were the same as those used in the MERIT trial (Ziebe et al.,2007).Secondary end-points included follicular development, number of oocytes retrieved, number of oocytes, fertilization, fertilization rate, number of embryos transferred, implantation rate, duration of stimulation, total dose of rFSH, serum levels of endocrine parameters, pregnancy rates and miscarriage rates.Result: Primary endpoint:The number of top-quality embryos per patient was analysed as a Poisson-distributed count. The mean numbers of top-quality embryos was4.44±3.79in study group, with a statistically significant higher than in control group3.14±2.46,(t=-2.028,P=0.045).Secondary end-pointsClinical outcomes:1. The total dose of rFSH consumed was significantly lower in the hCG group:2348±726IU versus2688±777IU,(t=2.26,P=0.026)。2. The number of oocytes retrieved, number of M2oocytes and fertilization were10.26±6.72,9.26±6.05, and6.78±5.17in hCG group,9.08±6.08,8.04±5.46and5.56±3.94。There were no significant differences between two group.3. The number of transferable embryos, transferal embryo and frozen embryos in study group were6.18±4.76,2.14±0.65and4.64±5.18. The corresponding number in control group were4.64±3.51,2.00±0.46and3.06±3.55in control group。Although the level was higher in study group, but no significant differences.4. The implantation rate, pregnancy rate and miscarriage rate, ongoing pregnancy rate were comparable in both groups. P>0.05.Serum endocrinology1. Estradiol:The FSH with low dose hCG group had higher peak estradiol level (4112±1186Vs3097±1566pg/mL, p<0.01,t=-3.65) than the FSH only group.2. Progesterone:There were no significant differences between two group in P level on HCG day. Although which was1.65±0.96mg/ml in study group, higher than in control group1.40±0.62mg/ml，p=0.13.3. LH level:The LH level on HCG triggering day was1.70±1.02mIU/mL in study group, which is lower than in control group2.9±3.03mIU/mL, p=0.006。4. HCG level:Steady state level of s-hCG was reached8.77IU on Day6of stimulation in study group.Adverse reactions events OHSS was not seen in study group, however, one patient in control group was diagnosed with morderate OHSS (outpatient).Summary:1. Supplementation with hCG from the first day of stimulation in GnRH antagonist protocol may increase the number of top-quality embryos.2. Supplementation with hCG from the first day of stimulation in GnRH antagonist protocol has the advantage of decreasing the dose of FSH and no obvious side effect, which is an new alternative COH protocol.Part two Human chorionic gonadotropin from embryo culture media and its relationship to embryo developmentMaterials and methodsPatients and treatment protocol:same in part oneSpent Embryo culture Media collection:A total of178day3spent embryo culture media from the patient IVF treatment, which inclusion in part one. The Day3embryos were transferred into uterus. The spent culture media were collected into eppendorf tube within15minutes, which was kept frozen individually in labeled microcentrifuge vials at--80℃until analysis. A sample of pure clture media incubated under the same condiction but without an embryo was also kept frozen and used as a blank control.HCG determination by ELISAA quantitative sandwich ELISA was developed in house and was performed following the optimization on spent embryo culture media. RayBio Amercian.Embryo Development monitoringThe embryo quality evaluation consisted of assessment of cell number and three parameters of embryo morphology, degree of fragmentation.Following the pregnancy rate and implantation rate Study end-pointsThe primary end-point evaluated the relationship of HCG value in day3embryo culture with the embryo development, pregnancy outcome, and implantation rate.Secondary end-point compared the HCG value in day3embryo culture in low-dose HCG group and rFSH group.Result:1. Of165spent embryo culture,10had no detectable amounts of hCG15were control samples, which include saline, Cleavage liquid and no embryo control sample. The value of hCG in control samples were lower0.1mIU/ml.75samples were from study group low-dose HCG group and78samples were from FSHgroup.2. The value of hCG in embryo culture between two group:In study group0.93±0.25mIU/ml, significant higher than FSH group0.81±0.21mIU/ml。 t=-2.39, p=0.02。3. The concentration of hCG in culture media increased gradually along with the increase number of blastomeres and decreased with the morphology grade. F=2.273, p=0.03and F=3.900, P=0.024. The amount of hCG correlated positively with implantation rate in both group. F=4.07, p=0.0155. The relationship between pregnancy outcome and hCG value in embryo culture: A total of74patients underwent embryo-transfer procedure. In FSH group, hCG value was higher in pregnancy group0.88±0.18IU than in non-pregnancy group0.74±0.21IU。t=-2.15, p=0.038.Summary:1. ELISA may be an optimal choice for detecting hCG in D3spent culture media.2. The concentration of hCG in spent culture media was correlated positively with embryo development monitoring, implantation rate, which may be as a useful marker for embryo selection in IVF-ET procedure. 3. The concentration of hCG in D3spent culture media was increase in the group which addition hCG in COH procedure.Part Three LH/HCG receptor protein expression in blastomere from day3human embryoMaterials and methodsPatients and treatment protocol:same in part oneInstitutional approval and informed consent For both ethical and practical reasons, this investigation was performed on normal fertilized but discarded embryos due to poor embryo score in day3embryo.Cleavage stage embryo biopsy and hCG-receptor FluorescenceImmunocytochmistry on blastomeres Preparation:D3discarded embryos for both experimental and control groups were collected and cryopreserved for test. Before Fluorescencelmmunocytochmistry to be performed, all of the embryos were thawed and cultured in blastycyst medium at370C and6%CO2for at lest1hr. Operation dishes containing Ca/Mg free HEPES buffered HTF droplet covered with oil were prepared a half hour before biopsy operation and warmed in desktop incubator, without CO2gas. Holding and biopsy needles were fixed on micro-operating system, adjusted to horizontal position and opposite direction, and elevated to the level under which operation dishes can easily put on the objective stage. Blastomere washing dishes with PBS droplets were prepaired just by blastomere fixation.Biopsy:Transfer each embryo to be biopsied into the individual droplet labeled with corresponding serial number, and put the dish on the objective stage. Lower the needles and flush them in the droplet without embryo. Locate and aspriate to fix the embryo on the top of the holding needle in the position with maxmal perivitelline space is opposite, considering minimizing the heat damage of laser to blastomere. Punch a hole with laser in a minimum diameter about30-40uM for the biopsy needle to enter and aspirate the each blastomeres gently out succesively.Blastomere fixation:First transfer blastomeres to droplets in the washing dishes and flush intensively, and then transfer to slides within the circle made by marker pen, naturally dried in room temperature.Fluorescence Immunocytochmistry on blastomeres Immunochemistry Blue fluorescent DAPI tags blatomere nuclei. Immunochemistry red fluorescent tags HCG receptor in blstomere.(DyLight(?)594).Collect the clinical data and following the pregnancy outcomeResult:Total40discarded D3embryos were collected from32patients. There were13embryos failed to perform immunohistochemical staining due to cell dropping out during the procedure. There were27embryos complete the procedure.7embryos were from the low-dose HCG group and20embryos were from rFSH group.Immunohistochemical StudyImmunohistochemical localiztiong of LH/HCG receptor showed intense staining of the different blastomere at Day3embryo. Higher expression on membrane than on cytoplasm..The HSCORE for LH/HCG receptor staining was higher in low-dose HCG group (1.793±0.210)than in rFSH group(1.394±0.318), t=-3.073, P=0.005Summary:1. Blatomere biopsy plus immunohistochemical localization of Lh/HCG receptors showed intense staining in the plasma membrane in all the blstomere at day3embryos, cytoplasm also low express.2. The higher expression of Lh/HCG receptor in D3embryo in the group which addition hCG in COH procedure. But the sample size was limited. Conclusion:1. Supplementation with hCG from the first day of stimulation in GnRH antagonist protocol may increase the number of top-quality embryos.2. ELISA may be an optimal choice for detecting hCG in D3spent culture media. The concentration of hCG in spent culture media was correlated positively with embryo development monitoring, implantation rate, which may be as a useful marker for embryo selection in IVF-ET procedure.3. Blatomere biopsy plus immunohistochemical localization of LH/HCG receptors showed intense staining in the plasma membrane in all the blstomere at day3embryos.4. The concentration of hCG in D3spent culture media and the expression of Lh/HCG receptor in D3embryo were increase in the group which addition hCG in COH procedure.5. Supplementation with hCG from the first day of stimulation in GnRH antagonist protocol has the advantage of decreasing the dose of FSH and no obvious side effect, which may be a new alternative COH protocol. |
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