L.C. Krey

Factors useful in predicting the success of oocyte donation: a 3-year retrospective analysis

OBJECTIVE To establish prognostic relevance of parameters assessed in oocyte donation cycles. DESIGN Retrospective analysis. SETTING Large university-based donor oocyte program. PATIENT(S) All oocyte recipient cycles achieving embryo transfer from September 1995 to October 1998. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Pregnancy. RESULT(S) Recipient age and reproductive status, day 9 and 12 serum estradiol (E(2)) levels and a progesterone (P) level obtained 2 days after initiation of hormonal therapy did not correlate with pregnancy. Endometrial thickness, but not endometrial pattern, was useful in predicting pregnancy outcome. The clinical pregnancy and live-birth rate in cycles where the endometrial thickness was less than 8 mm was significantly lower when compared to cycles with an endometrial thickness > or =9 mm. Cycles where optimal quality embryos were transferred had the highest implantation (36%), clinical pregnancy (63%) and live birth (54%) rates and these rates were significantly higher than those of cycles where only poor quality embryos were available for transfer (10% implantation, 17% clinical pregnancy, and 8% live birth rates, respectively; P<.05). CONCLUSION(S) The most reliable predictive factors for pregnancy in oocyte donation cycles are the quality of the embryos transferred and the recipients mid-cycle endometrial thickness. Recipient monitoring should minimally include ultrasound assessment of endometrial thickness.

Monozygotic twinning: an eight-year experience at a large IVF center

OBJECTIVE To characterize incidence, chorionicity, amnionicity, and pregnancy outcome for monozygotic twin pregnancy (MZT) after IVF. DESIGN Retrospective review. SETTING University-based fertility center. PATIENT(S) Autologous and oocyte donation IVF cycles eventuating in 4,976 clinical gestations from 2000 to 2007. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) MZT incidence, chorionicity, zygosity, pregnancy outcome. RESULT(S) Ninety-eight MZTs were diagnosed after first-trimester ultrasound evaluation (2% incidence). The incidence in cycles transfering autologous oocytes was 1.7% but was 3.3% with donor oocytes; however, women <35 years old using their own oocytes displayed a similar rate (3.1%) to women using donor oocytes. Eighty MZTs occurred after fresh day-5 transfer; only 14 followed fresh day-3 transfer (2.6% vs. 1.2%). The MZT incidence in day-3 transfers without hatching was not different from those with hatching (1.3% vs. 1.1%). In addition, MZT incidence did not differ significantly whether or not ICSI was performed (2.4% vs. 2.0%). Four MZTs occurred after frozen-thawed embryo transfer (0.8% incidence). Ninety-five percent of all placental arrangements were confirmed as monochorionic-diamniotic on obstetric ultrasounds. CONCLUSION(S) These findings confirm a higher incidence of MZT after IVF. Monochorionic-diamniotic implantations were increased, whereas monochorionic-monoamniotic were not. The MZT risk factors included young age and extended culture, but not zona penetration or cryopreservation.

Women with cancer undergoing ART for fertility preservation : a cohort study of their response to exogenous gonadotropins

Cancer patients produce similar numbers of oocytes after ovarian hyperstimulation compared with age-matched infertile controls, suggesting that malignancy does not adversely affect ovarian response.

Enhancement or initiation of testicular sperm motility by in vitro culture of testicular tissue

OBJECTIVE To describe different techniques of testicular tissue culture and their effect on sperm motility, mainly in cases of totally immotile spermatozoa, and to compare the effect of in vitro culture with that of motility stimulants. DESIGN Prospective study. SETTING University teaching hospital. PATIENT(S) Ten patients undergoing testicular biopsy for diagnostic purposes or for intracytoplasmic sperm injection. INTERVENTION(S) Dissected testicular biopsy samples and tissue blocks were cultured at 37 degrees C for up to 96 hours. Immediately after dissection, immotile testicular spermatozoa were incubated for 30 minutes in pentoxifylline and 2-deoxyadenosine. MAIN OUTCOME MEASURE(S) Sperm motility and vitality. RESULT(S) Overall, dissected samples showed improved sperm motility, which peaked within 48 hours of culture. Unlike motility, vitality declined linearly, from 56.3%+/-19% at initial assessment to 18.8%+/-11% at 96 hours. Five samples had initially immotile spermatozoa, of which four acquired motility at 48 hours. In vitro culture showed results comparable with those of incubation with pentoxifylline and 2-deoxyadenosine. Culture of tissue blocks did not improve motility or vitality compared with dissected tissue. CONCLUSION(S) The motility of testicular spermatozoa was enhanced or initiated after in vitro culture. Testicular biopsy culture may be an alternative to the use of motility stimulants to obtain motile spermatozoa for intracytoplasmic sperm injection, particularly when oocytes are not immediately available.

Simultaneous assessment of sperm chromatin condensation and morphology before and after separation procedures: effect on the clinical outcome after in vitro fertilization

OBJECTIVE To look for correlations between acridine orange (AO) staining and semen parameters before and after sperm separation procedures and to assess whether the AO test predicts fertilization or pregnancy outcomes after standard IVF and intracytoplasmic sperm injection. DESIGN Prospective study that simultaneously assesses sperm morphology and nuclear protein maturity on a cell-by-cell basis before and after preparative procedures. SETTING University teaching hospital. PATIENT(S) Men (n = 140) undergoing diagnostic semen analysis. MAIN OUTCOME MEASURE(S) Acridine orange fluorescence of sperm nuclei, semen parameters, IVF outcome. RESULT(S) In unprocessed samples, 90% of sperm with normal heads displayed green fluorescence (mature nuclear protein); significantly lower percentages of green fluorescence were observed in sperm with abnormal heads. The percentage of mature normal sperm in the specimen correlated with motility. Sperm maturity after swim-up or Percoll gradient was significantly improved for sperm with normal or abnormal heads. The percentage of mature normal sperm correlated with motility after either Percoll or swim-up. Neither the percentages of mature nuclei nor mature normal nuclei correlated with fertilization or pregnancy outcome. CONCLUSION(S) Nuclear protein maturation correlates with sperm motility and morphology. Because morphologically normal and motile sperm are more mature, separation procedures should generate a population of sperm with the highest fertilization capacity. Acridine orange staining, however, did not predict fertilization efficiency or pregnancy outcome in IVF cycles.

Preimplantation genetic diagnosis in two families at risk for recurrence of Herlitz junctional epidermolysis bullosa

Abstract: The Herlitz type of junctional epidermolysis bullosa (H‐JEB) is a severe inherited bullous disease which leads to the early demise of the affected newborn. Mutations in the genes encoding the 3 polypeptides of the anchoring filament protein laminin 5 underlie this condition. We studied 2 families with affected children who previously died from H‐JEB. Mutation screening using heteroduplex analysis and direct sequencing of the PCR products revealed a previously described hotspot mutation in LAMB3 (R635X), and a novel delayed termination codon in LAMB3 in the first proband. In the second proband, we found a novel initiation codon mutation in LAMB3, and a novel 2 bp deletion in LAMB3. For preimplantation genetic diagnosis (PGD) in these families, we developed nested multiplex PCR assays, amplifying the mutations and informative intragenic polymorphisms in the probands. Single embryonic cells were biopsied from 8‐cell embryos using standard techniques, and subjected to the multiplex PCR assay followed by restriction enzyme digestion. Embryos found not to carry either mutation were transferred to the mothers, and a pregnancy was established in the second family as evidenced by the elevated level of HCG, although the pregnancy did not persist. This study illustrates the feasibility of PGD for an inherited skin disorder for the first time.

Candidate lineage marker genes in human preimplantation embryos

Cell allocation and subsequent lineage commitment in the human embryo may be established as early as in the unfertilized oocyte. This phenomenon might be the result of subtle differences of gene expression and protein distribution. To assess whether gene expression profiling by reverse transcription-polymerase chain reaction could be a suitable tool for the detection of cell allocation and lineage commitment, the expression pattern of the putative inner cell mass marker gene Oct-4 and the trophectodermal marker genes beta-HCG and beta-LH were correlated in individual blastomeres of preimplantation human embryos. In 2- to 5-cell stage embryos, expression of beta-HCG and Oct-4 mRNA was negatively correlated in all blastomeres with statistical significance, suggesting that cell allocation can be assessed by those markers at early stages. In 7- to 10-cell stage embryos, expression of beta-LH and Oct-4 mRNA was negatively correlated in some blastomeres without statistical significance, suggesting that more experiments are necessary to decide if lineage commitment can be assessed in some cells by those markers at later stages.

The antiviral agents, MAP30 and GAP31, are not toxic to human spermatozoa and may be useful in preventing the sexual transmission of human immunodeficiency virus type 1

OBJECTIVE To investigate the effects of two virucidal compounds, MAP30 (Momordica anti-human immunodeficiency virus [HIV] protein; molecular weight, 30 kd) and GAP31 (Gelonium anti-HIV protein; molecular weight, 31 kd), obtained from Momordica charantia and Gelonium multiflorum, respectively, on the motility and vitality of human spermatocytes. DESIGN Prospective, controlled study. SETTING New York University School of Medicine. PATIENT(S) Ten healthy men undergoing evaluation for infertility provided 10 semen specimens. INTERVENTION(S) Human sperm were treated with the anti-HIV agents, MAP30 and GAP3 1. Nonoxynol-9, a commonly used spermicide, and phosphate-buffered saline were used as the positive and negative controls, respectively. MAIN OUTCOME MEASURE(S) The motility and vitality of human spermatocytes treated with MAP30 and GAP31 at doses that inhibit HIV-1 and herpes simplex virus. RESULT(S) MAP30 and GAP31 did not inhibit the motility or vitality of human sperm cells over a dose range of 100-0.1 microg/mL, whereas nonoxynol-9 demonstrated spermicidal action on all 10 samples over the same dose range. CONCLUSION(S) The antiviral agents, MAP30 and GAP31, were not toxic to human sperm cells at the doses at which they inhibit HIV-1 and herpes simplex virus. They had no effect on the motility of spermatozoa, even at a dose of 1,000 times the maximum effective concentration. These results indicate that MAP30 and GAP31 may be useful as nonspermicidal protection against sexually transmitted diseases.

Assessment of β-HCG, β-LH mRNA and ploidy in individual human blastomeres

In human embryos, blastomeres differentiate into trophectoderm (TE) cells and inner cell mass (ICM) cells of blastocysts. Although morphologically indistinguishable, blastomeres at early cleavage stages are likely to undergo changes on a molecular level that make them destined to become ICM or TE cells. While the transcription factor Oct-4 might serve as a marker for totipotent ICM cells, human chorionic gonadotrophin might be used as the equivalent for TE cells. This study reports a reverse transcription-polymerase chain reaction procedure to assess human β-HCG mRNA concentrations as well as ploidy in individual blastomeres from normally and abnormally fertilized human embryos. β-HCG mRNA was detected in both euploid and aneuploid cells and in oocytes. Surprisingly, β-LH mRNA was also detected in some euploid blastomeres. In regard to preimplantation genetic diagnosis, assessment of expression levels of β-HCG and Oct-4 mRNA in individual biopsied cells might serve as a tool to identify embryogenic blastomeres in combination with testing for chromosome and single gene abnormalities.

Preimplantation Genetic Diagnosis of Human Embryos for Marfan's Syndrome

Purpose:Single-cell nested polymerase chain reaction (PCR) and Dde1 endonuclease digestion were used to detect the presence of a Marfans syndrome mutation in human preimplantation embryos derived from in vitro fertilization (IVF). These procedures were conducted to eliminate the possibility of transmission of the affected allele from the father to his offspring. The mutation on chromosome 15 is transmitted as an autosomal dominant trait, and the chance of having a child affected with the disease is 50%.Methods:A couple presented to the Program for In Vitro Fertilization, Reproductive Surgery and Infertility for preimplantation genetic diagnosis. IVF was performed and embryo biopsy was done on day 3 embryos, Single blastomeres were removed from embryos and subjected to nested PCR analysis and endonuclease digestion to detect a Marfans syndrome mutation located on chromosome 15 inherited from the father.Results:Thirteen oocytes were injected with spermatozoa using intracytoplasmic sperm injection, and nine fertilized normally. Following embryo biopsy and polymerase chain reaction amplification-Dde1 endonuclease digestion, five embryos were detected that were positive for the mutation. The four non-affected embryos were transferred to the uterus, resulting in a healthy and normal ongoing pregnancy.