Guanglun Zhuang

High follicle-stimulating hormone increases aneuploidy in human oocytes matured in vitro

OBJECTIVEnTo study the effect of FSH on the aneuploidy risk of human oocytes matured in vitro.nnnDESIGNnProspective study.nnnSETTINGnHospital-based IVF center.nnnPATIENT(S)nPatients with male factor infertility undergoing intracytoplasmic sperm injection (ICSI) cycles.nnnINTERVENTION(S)nImmature oocytes were put into five groups according to the FSH concentration (0, 5.5, 22, 100, and 2,000 ng/mL) in in vitro maturation (IVM) medium. Spindles were observed under a polarized microscope before polar body biopsy. Fixed polar bodies and corresponding oocytes were examined on chromosomes 13, 16, 18, 21, and 22 by fluorescence in situ hybridization. Oocytes matured in 5.5 and 2,000 ng/mL FSH were immunostained for tubulin and chromatin.nnnMAIN OUTCOME MEASURE(S)nAneuploidy rate, spindle visualization rate, and spindle morphology.nnnRESULT(S)nThe frequency rates of aneuploidy were 26.7%, 23.3%, 36.75%, 46.67%, and 63.3% in the five FSH groups, respectively. There was a significantly higher aneuploidy rate in oocytes matured in the 2,000 ng/mL FSH group. The spindle visualization rates assessed under PolScope were not significantly different between aneuploid and normal oocytes. There was no difference in spindle morphology between the 2,000 and 5.5 ng/mL FSH groups.nnnCONCLUSION(S)nHigh-concentration FSH in IVM medium significantly increased the first meiotic division error, resulting in more aneuploid oocytes during IVM.

Pregnancy derived from human zygote pronuclear transfer in a patient who had arrested embryos after IVF

Nuclear transfer of an oocyte into the cytoplasm of another enucleated oocyte has shown that embryogenesis and implantation are influenced by cytoplasmic factors. We report a case of a 30-year-old nulligravida woman who had two failed IVF cycles characterized by all her embryos arresting at the two-cell stage and ultimately had pronuclear transfer using donor oocytes. After her third IVF cycle, eight out of 12 patient oocytes and 12 out of 15 donor oocytes were fertilized. The patients pronuclei were transferred subzonally into an enucleated donor cytoplasm resulting in seven reconstructed zygotes. Five viable reconstructed embryos were transferred into the patients uterus resulting in a triplet pregnancy with fetal heartbeats, normal karyotypes and nuclear genetic fingerprinting matching the mothers genetic fingerprinting. Fetal mitochondrial DNA profiles were identical to those from donor cytoplasm with no detection of patients mitochondrial DNA. This report suggests that a potentially viable pregnancy with normal karyotype can be achieved through pronuclear transfer. Ongoing work to establish the efficacy and safety of pronuclear transfer will result in its use as an aid for human reproduction.

Preimplantation genetic diagnosis for Duchenne muscular dystrophy by multiple displacement amplification

OBJECTIVEnTo evaluate the use of multiple displacement amplification (MDA) in preimplantation genetic diagnosis (PGD) for female carriers with Duchenne muscular dystrophy (DMD).nnnDESIGNnMDA was used to amplify a whole genome of single cells. Following the setup on single cells, the test was applied in two clinical cases of PGD. One mutant exon, six short tandem repeats (STR) markers within the dystrophin gene, and amelogenin were incorporated into singleplex polymerase chain reaction (PCR) assays on MDA products of single blastomeres.nnnSETTINGnCenter for reproductive medicine in First Affiliated Hospital, Sun Yat-sen University, China.nnnPATIENT(S)nTwo female carriers with a duplication of exons 3-11 and a deletion of exons 47-50, respectively.nnnINTERVENTION(S)nThe MDA of single cells and fluorescent PCR assays for PGD.nnnMAIN OUTCOME MEASURE(S)nThe ability to analyze single blastomeres for DMD using MDA.nnnRESULT(S)nThe protocol setup previously allowed for the accurate diagnosis of each embryo. Two clinical cases resulted in a healthy girl, which was the first successful clinical application of MDA in PGD for DMD.nnnCONCLUSION(S)nWe suggest that this protocol is reliable to increase the accuracy of the PGD for DMD.

Mechanically expanding the zona pellucida of human frozen thawed embryos: a new method of assisted hatching

OBJECTIVEnTo determine whether a new assisted hatching (AH) method increases the implantation and clinical pregnancy rates of frozen-thawed day-3 (D3) embryos.nnnDESIGNnProspective study.nnnSETTINGnA university hospital in vitro fertilization (IVF) program.nnnPATIENT(S)nPatients who had their first IVF/intracytoplasmic sperm injection (ICSI) cycles between June 1, 2006, and December 31, 2008, with fresh IVF-embryo transfer failures or without fresh embryo transfer.nnnINTERVENTION(S)nThe couples were randomized into thawed embryo transfer after AH versus no AH. In the AH group, the zona pellucida (ZP) of D3 frozen-thawed embryos was expanded by injected hydrostatic pressure after thawing. In the control group, embryos were pierced by ICSI needles without expanding the ZP.nnnMAIN OUTCOME MEASURE(S)nClinical pregnancy and implantation rates.nnnRESULT(S)nThe morphologic features of the blastomeres were carefully monitored and recorded. In the AH group, 244 embryos were thawed, and 178 (73.0%) survived; in the control group, 259 embryos were thawed, and 190 (73.4%) survived. Despite the transfer of a similar number of embryos, the AH group resulted in statistically significantly higher implantation and clinical pregnancy rates compared with the no AH group.nnnCONCLUSION(S)nMechanically expanding the ZP of frozen-thawed D3 embryos with injected hydrostatic pressure after thawing increases the implantation rate compared with control embryos.

Preimplantation genetic diagnosis for α-and β-double thalassemia

PurposeTo evaluate the use of multiple displacement amplification (MDA) for preimplantation genetic diagnosis (PGD) of α- and β-double thalassemia.MethodWhole genome of a single cell was directly amplified using MDA and its products were used as templates in fluorescent gap polymerase chain reaction (PCR) analysis of α-thalassemia and in PCR-reverse dot blot analysis ,singleplex fluorescent PCR of β-28 and CD17 mutation and HumTH01 for β-thalassemia.Results1) MDA from single cell could produce enough DNA templates for the detection of both α and β-thalassemia; 2) The established MDA-PGD protocol for α- and β-double thalassemia was successfully applied in PGD of six embryos, among which, three were transferred, but no pregnancy ensued.ConclusionsThe use of MDA as a universal step allows for the simultaneous diagnosis of two or more hereditary defects.

Preimplantation genetic diagnosis for α-thalassaemia in China

PurposeTo report the usage of PGD for α-thalassaemia with the -u2009-SEA genotype.MethodA PGD protocol using fluorescent gap PCR was performed for 51 cycles on 43 couples with the -u2009-SEA genotype. Allele drop-out and amplification failure rates were retrospectively analyzed.ResultsA total of 472 embryos were biopsied. Amplification was achieved in 390 blastomeres, accounting for an amplification rate of 82.6%. In total, 120 wild-type, 94 heterozygotes and 140 homozygous mutant embryos were diagnosed. The successful diagnosis rate was 75.0%. The ADO rate in 49 blastomeres from six donated embryos was 16.4%. One hundred and fifty four embryos were transferred, resulting in 25 clinical pregnancies with an implantation rate of 24.0%.ConclusionsSingle-round fluorescent gap PCR is a feasible and effective strategy in the PGD for α-thalassaemia with the -u2009-SEA genotype.

Expression of certain HLA-I types in cleavage-stage embryos

This study examined the expression of human leukocyte antigen (HLA)-G and HLA-I (which includes HLA-A, -B, -C, -E and -F, but is without HLA-G) in the cleavage embryo and its supernatant, and related the results to embryo development including growth rate and grade. In total, 136 day-3 cleavage embryos were used for detection of HLA-G and 24 embryos for HLA-I without HLA-G by immunohistochemistry. The expression of HLA-I was examined by western blot in the lysates of a further 63 day-3 cleavage embryos; soluble HLA-I in the culture supernatant of embryos with detectable HLA-I was also examined by western blot. It was found that 90 of 136 (66.2%) cleavage embryos expressed HLA-G, whereas 23 of 24 (95.8%) embryos expressed HLA-I without HLA-G. HLA-G expression typically showed an even and symmetrical pattern of distribution in each blastomere. HLA-I without HLA-G in cleavage-stage embryos is typically scattered around the blastomere surface. The expression of HLA-G but without HLA-I in cleavage-stage embryos was significantly associated with embryo grade (P < 0.001) and cell number (P = 0.03). In conclusion, HLA-I is expressed on day-3 cleavage embryos, and HLA-G expression on preimplantation embryos is related to embryo development, including embryo growth rate and embryo grade.

Supraphysiological estrogen levels adversely impact proliferation and histone modification in human embryonic stem cells: possible implications for controlled ovarian hyperstimulation assisted pregnancy

OBJECTIVEnControlled ovarian hyperstimulation (COH) results in supraphysiologic levels of maternal serum estradiol (E(2)) during the luteal phase, thus promoting oocyte production at unknown risk to the subsequently developing embryo. Human embryonic stem cells (hESCs) have been identified as a model system to assess the impact of COH on early embryonic development, specifically 17β-estradiol mediated effects on proliferation, gene expression, and histone modification.nnnSTUDY DESIGNnCell proliferation and associated factors, such as HDAC1, as well as histone modification patterns were evaluated in ERα and β expressing hESCs after exposure to 17β-estradiol (1×10(-10) M to 1×10(-7) M), as well as in an untreated control.nnnRESULTSnResultant data revealed that while physiologically relevant E(2) levels (1×10(-9)M E(2)) induced cell cycle progression from G1 to the proliferation phase, supraphysiologic levels akin to those observed after COH (1×10(-7) M E(2)) adversely affected hESCs proliferation via down regulation of HDAC1. Modification of H3K9me2, PhH3S10, H4K5ac, and H2A.Z histone patterns were also dependent on 17β-estradiol concentration.nnnCONCLUSIONnWhile physiologic levels of 17β-estradiol induced cell proliferation, possibly via HDAC1 involvement in histone modification, cell proliferation in hESCs was suppressed at supraphysiologic levels.

Impact of Transitory Hyperprolactinemia on Clinical Outcome of In Vitro Fertilization and Embryo Transfer

Impact of Transitory Hyperprolactinemia on Clinical Outcome of In Vitro Fertilization and Embryo Transfer This study aimed to evaluate the impact of serum prolactin concentration at the day of human chorionic gonadotropin (HCG) administration on the clinical outcome of in vitro fertilization and embryo transfer (IVF-ET). A total of 184 patients receiving the IVF-ET/ICSI-ET from October 2005 to March 2008 were retrospectively analyzed. Subjects were divided into four groups according to the serum prolactin concentration [<30 ng/mL (A), 30-60 ng/mL (B), 60-90 ng/mL (C), ≥90 ng/mL (D)] on the day of HCG administration during controlled ovarian stimulation (COS). In the Groups A, B, C and D, the implantation rate was 11.76%, 19.71%, 12.72% and 2.22%, respectively, and the pregnancy rate (PR) was 25.00%, 42.70%, 27.30% and 5.88%, respectively. The implantation rate and PR in the Group D were markedly lower than those in the remaining groups (P=0.011 and 0.009). During the COS, the serum prolactin concentration was dramatically elevated when compared with the baseline level leading to transient hyperprolactinemia. In addition, the implantation rate and pregnancy rate were significantly markedly decreased when the serum prolactin concentration was remarkably increased (≥90 ng/mL). To improve the clinical pregnancy rate of IVF-ET, close monitoring and appropriate intervention are needed for patients with an abnormal prolactin level during the COS. Uticaj Prolazne Hiperprolaktinemije Na Klinički Ishod Veštačke Oplodnje i Transfer Embriona Ova studija sprovedena je kako bi se procenio uticaj koncentracije prolaktina u serumu na dan administracije humanog horionskog gonadotropina (HCG) na klinički ishod in vitro oplodnje i transfera embriona (IVF-ET). Retrospektivna analiza je obuhvatila ukupno 184 pacijenta podvrgnuta IVF-ET/ICSI-ET između oktobra 2005. i januara 2008. Ispitanici su podeljeni u četiri grupe na osnovu koncentracije prolaktina u serumu [<30 ng/mL (A), 30-60 ng/mL (B), 60-90 ng/mL (C), ≥90 ng/mL (D)] na dan administracije HCG tokom kontrolisane stimulacije jajnika (COS). U grupama A, B, C i D stopa implantacije bila je 11,76%, 19,71%, 12,72% i 2,22%, dok je stopa ostvarenih trudnoća (PR) bila 25,00%, 42,70%, 27,30% i 5,88%. Stopa implantacije i PR u grupi D bile su upadljivo niže nego u ostalim grupama (P=0,011 i 0,009). Tokom COS, koncentracija prolaktina u serumu bila je dramatično povišena u poređenju sa početnim nivoom, što je dovelo do prolazne hiperprolaktinemije. Pored toga, stopa implantacije i stopa ostvarenih trudnoća bile su značajno i upadljivo niže kada je koncentracija prolaktina u serumu bila izrazito povišena. Pažljivo praćenje i odgovarajuće intervencije potrebni su kako bi se poboljšala klinička stopa ostvarenih trudnoća u IVF-TE kod pacijenata sa abnormalnim nivoom prolaktina tokom kontrolisane stimulacije jajnika.