Asangla Ao

Production of steroids from human cumulus cells treated with different concentrations of gonadotropins during culture in vitro

OBJECTIVE To evaluate the output of E2 and progesterone produced by cumulus cells, derived from mature and immature oocytes, in culture medium. DESIGN Prospective randomized study. SETTING McGill Reproductive Center, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada. PATIENT(S) Twenty-one women, <38 years of age and with normal menstrual cycles, who were undergoing intracytoplasmic sperm injection for assisted reproduction. INTERVENTION(S) Culture medium with or without fetal bovine serum (FBS) supplemented with either a physiologic (75 mIU/mL) or a supraphysiologic (7,500 mIU/mL) concentration of gonadotropins. MAIN OUTCOME MEASURE(S) Comparison of steroid levels in culture medium. RESULT(S) Estradiol secretion was significantly increased in the culture medium with FBS supplemented with both concentrations of FSH alone compared with control. However, E2 secretion was inhibited by both concentrations of FSH with LH. The level of E2 was undetectable in the medium without FBS even after supplementation with both concentrations of FSH alone, hCG alone, and FSH with LH. Progesterone production was increased in the medium with FBS supplemented with FSH alone, hCG alone, and FSH with LH compared with control. There was no difference in progesterone levels in the culture medium without FBS supplemented with both concentrations of FSH alone and hCG alone compared with control. However, progesterone secretion was increased in the medium without FBS supplemented with a physiologic concentration of FSH with LH. CONCLUSION(S) Culture medium with FBS supplemented with a physiologic and a supraphysiologic concentration of FSH stimulates E2 secretion from cumulus cells derived from mature and immature oocytes. This suggests that it may be not necessary to add E2 to the culture medium for maturation in vitro of immature human oocytes retrieved from patients undergoing stimulated cycles.

High rate of mixoploidy among human blastocysts cultured in vitro

OBJECTIVE To determine the incidence and type of mixoploidy in human blastocysts produced in vitro. DESIGN A laboratory study of spare blastocysts from an IVF program. SETTING University hospital laboratory. PATIENT(S) Thirty-nine couples undergoing IVF or intracytoplasmic sperm injection. INTERVENTION(S) A total of 103 blastocysts were classified as good- or poor-quality blastocysts based on morphology. A total of 6,927 interphase nuclei, 5,015 from 59 good-quality and 1,912 from 44 poor-quality blastocysts, were assessed for ploidy by fluorescence in situ hybridization with chromosome-specific DNA probes. MAIN OUTCOME MEASURE(S) The percentage and the type of polyploid cells present in each blastocyst. RESULT(S) Mixoploidy (mixture of diploid and polyploid cells) was found in 86% of good-quality and 82% of poor-quality blastocysts analyzed. The type of polyploidy ranged from 3N to 14N, with tetraploidy being the most common between both groups. The proportion of polyploid cells per mixoploid blastocyst ranged from 1% to 88%. The percentage of polyploid nuclei within most good-quality mixoploid blastocysts was small (10%) and significantly lower than in poor-quality blastocysts (19%). CONCLUSION(S) Most human blastocysts produced in vitro contain polyploid, predominantly tetraploid cells. The proportion of polyploid cells in the majority of good-quality blastocysts is low. The small numbers of blastocysts with a high percentage of polyploid cells may have implications for blastocyst transfer.

Chromosome abnormality rates in human embryos obtained from in-vitro maturation and IVF treatment cycles

The aim of this retrospective study was to compare the incidence of chromosomal abnormality in embryos from in-vitro maturation (IVM) and IVF cycles. The copy numbers of chromosomes 13, 15, 16, 18, 21, 22, X and Y were assessed with fluorescence in-situ hybridization (FISH) in single blastomeres biopsied from cleavage stage embryos. Spare embryos that were not transferred or cryopreserved were also analysed in full. IVM and IVF groups comprised six and 30 couples, with mean ± SD embryos with FISH result of 8.0 ± 4.4 and 11.7 ± 3.8, respectively. The incidence of chromosomal abnormality per FISH result was similar in IVM and IVF embryos (58.7% versus 57.4%, respectively). When embryos were categorized based on maturation time of oocytes in IVM cycles, embryos derived from oocytes that matured 48 h after collection had a higher chromosomal abnormality rate compared with embryos derived from in-vivo matured oocytes and to embryos derived from oocytes that matured in the first 24 h after collection.

Vascular endothelial growth factor A and its two receptors in human preantral follicles from fetuses, girls, and women

OBJECTIVE To investigate the expression of vascular endothelial growth factor A (VEGF-A) and that its two receptors (VEGFR1, VEGFR2) in human preantral follicles. DESIGN Immunohistochemical, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) study of the expression of the VEGF-A system in human ovaries. SETTING Major tertiary-care academic center. PATIENT(S) Twenty-two patients who underwent pregnancy terminations at 21-35 gestational weeks and 29 girls/women aged 5-39 years who underwent ovarian laparoscopies. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Laboratory analysis of human ovarian specimens. RESULT(S) Immunhistochemistry and in situ hybridization revealed the expression of the proteins and mRNA transcripts for VEGFR1 and VEGFR2 in oocytes, granulosa cells, and stroma cells from fetuses and girls/women. The protein for VEGF-A was detected immunohistochemically in oocytes, granulosa cells, and stroma cells from fetuses and girls. VEGF-A and VEGFR1 proteins were expressed more strongly than VEGFR2. VEGF-A(121), VEGF-A(165), and VEGF-A(189) isoforms were identified by RT-PCR in the ovarian samples from fetuses and women. CONCLUSION(S) The presence of the VEGF-A receptors, particularly in the granulosa cells, suggests that VEGF-A might be involved in proliferation initiation of primordial follicles or play an as yet unknown role in human preantral follicles.

Expression of growth-differentiating factor 9 and its type 1 receptor in human ovaries

The expression of growth-differentiating factor 9 (GDF9) has not been studied in ovaries from girls and human fetuses nor has its receptor transforming growth factor-beta1 receptor (TGFbetaR1) been investigated in ovaries of girls/women. The aim of this study was to fill these gaps. Ovarian samples were obtained from 16 human fetuses at 21-35 gestational weeks and from 34 girls/women aged 5-39years. These specimens were prepared for immunohistochemical staining of the GDF9 and TGFbetaR1 proteins. Reverse transcription polymerase chain reaction was used to detect GDF9 mRNA transcripts and in-situ hybridization to localize TGFbetaR1 mRNA transcripts. Positive staining for the GDF9 protein was identified in oocytes and granulosa cells in all samples tested. GDF9 mRNA transcripts were present in all samples. Protein expression of TGFbetaR1 was identified in granulosa cells in all samples. Oocyte staining was identified in samples from girls/women but in only one fetal sample. TGFbetaR1 mRNA transcripts were identified in granulosa cells and oocytes in 50% of the samples from fetuses aged over 22 gestational weeks and in samples from girls/women. The detection of GDF9 and TGFbetaR1 at both at the protein and mRNA levels suggests that GDF9 may have functions in human preantral follicles.

Glial cell line–derived neurotrophic factor (GDNF) and its receptors in human ovaries from fetuses, girls, and women

OBJECTIVE To investigate the expression of glial cell line-derived neurotrophic factor (GDNF) and its receptors, GDNF alpha 1 (GFR-alpha1) and tyrosine kinase receptor for rearranged during transfection (RET), in human ovaries at the protein and mRNA levels. DESIGN Samples were prepared for immunohistochemical staining for GDNF, GFR-alpha1, and RET and in situ hybridization for the mRNA of GFR-alpha1. The mRNA expression of two GDNF isoforms and two RET isoforms was investigated by reverse transcription polymerase chain reaction. SETTING Infertility unit at a university-affiliated tertiary medical center. PATIENT(S) Fifteen patients who underwent pregnancy terminations at 21-35 gestational weeks and 28 girls/women aged 5-39 years who underwent ovarian laparoscopies. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Laboratory analysis of human ovarian specimens. RESULT(S) The GDNF protein was detected in oocytes of all samples. Granulosa cells (GCs) stained for GDNF in a portion of fetal samples and in all samples from girls/women. The proteins for GFR-alpha1 and RET were detected in both oocytes and GCs with weak staining for the long RET isoform. GFR-alpha1 mRNA transcripts were detected in oocytes and GCs from all samples. The mRNA transcripts for the two GDNF isoforms and the two RET isoforms were detected in all fetal and adult specimens. CONCLUSION(S) The presence of the receptors of GDNF in the GCs of human primordial follicles suggests that GDNF may be involved in the regulation of primordial follicular activation.

Chromosomal complement and clinical relevance of multinucleated embryos in PGD and PGS cycles

The objective of this retrospective study was to investigate the incidence and clinical implications of multinucleation in blastomeres biopsied from cleavage-stage embryos obtained from patients undergoing preimplantation genetic screening (PGS) for aneuploidies or preimplantation genetic diagnosis (PGD) for translocations or single-gene defects (SGD). A total of 3515 embryos were obtained from 306 couples in 380 PGD or PGS cycles. Incidence of multinucleation, chromosomal complement in multinucleated (MN) and sibling embryos and the characteristics of MN embryos resulting in healthy births were investigated. Of all cycles, 41.3% involved at least one MN embryo. There were more uniformly diploid than uniformly haploid nuclei (22.0% versus 7.9%, P<0.01). The most common form of abnormality was chaotic chromosomal complement (39.9%, 147/368). Transfer of embryos that had MN blastomeres free of the genetic abnormalities tested resulted in three healthy deliveries. It is concluded that, although the majority of MN blastomeres are chromosomally abnormal, healthy births are possible after transfer of embryos containing these blastomeres subjected to genetic analysis. As far as is known, this is the first report of healthy births after transfer of embryos with MN blastomeres tested for translocations or SGD in PGD cycles. Preimplantation genetic diagnosis (PGD) is an established method for selecting genetically healthy embryos for transfer. A blastomere sampled from the developing embryo is subjected to genetic analysis. Some of these blastomeres may contain multiple nuclei, complicating the genetic diagnosis. We investigated clinical implications of multinucleation in PGD cycles. Our results indicate that majority of the multinucleated blastomeres, and consequently embryos, are genetically abnormal. However, healthy births are possible after transfer of multinucleated embryos that are free of the genetic abnormalities screened.

Chromosomal information derived from single blastomeres isolated from cleavage-stage embryos and cultured in vitro

OBJECTIVE To evaluate the potential of proliferation of single blastomeres isolated from human cleavage-stage embryos for use in preimplantation genetic diagnosis of chromosomal abnormalities. DESIGN A laboratory study of chromosomal content of blastomeres isolated from embryos of patients from an in vitro fertilization program. SETTING University hospital laboratory. PATIENT(S) Couples undergoing IVF or ICSI. INTERVENTION(S) Blastomeres were isolated from normally fertilized cleavage-stage human embryos, cultured in vitro or fixed immediately, and analyzed by fluorescence in situ hybridization (FISH) probes. MAIN OUTCOME MEASURES Chromosomal information yielded by blastomeres cultured in vitro compared with those obtained from blastomeres that were processed for chromosomal analysis directly after isolation. RESULT(S) The percentage of cultured blastomeres that produced FISH results was significantly lower than the percentage of blastomeres processed for FISH directly after isolation (72% vs. 90%). Lack of FISH results from cultured cells, which in most cases was related to nuclear anomalies, was significantly more frequent among nondivided than divided blastomeres (39% vs. 21%). Both cultured and noncultured cells showed diploid, aneuploid and polyploid chromosome complements on FISH. Compared with directly processed cells, cultured cells yielded a higher proportion of polyploid patterns (22.9% vs. 6.1%). Of the cultured blastomeres that divided, 18% produced progeny with mosaicism. CONCLUSION(S) Although blastomere culture may increase the number of cells available for chromosomal analysis, the high frequency of nuclear defects and the occurrence of polyploidy and mosaicism among cultured cells discourage the use of blastomere isolation and proliferation strategy for use in preimplantation genetic diagnosis.

Different probe combinations for assessment of postzygotic chromosomal imbalances in human embryos.

Purpose: We compared three different probe combinations for detection of postzygotic mosaic imbalances in human preimplantation embryos. Methods: Two hundred and two spare cleavage stage embryos were hybridized with fluorescently labelled DNA probe mixtures specific to chromosomes X, Y, 18 (N = 67), chromosomes 2, 7, 18 (N = 71), or chromosomes 13, 16, 18, 21, 22 (N = 64). Results: An overall higher incidence of abnormalities was detected using probe mixture for five (69%) or three (72%) autosomes compared to one autosome and chromosomes X and Y (54%). The rate of aneuploidy detected increased with the number of autosomes hybridized from 4% (X, Y, 18) to 11% (2, 7, 18) to 19% (13, 16, 18, 21, 22). Postzygotic mosaicism comprised the most frequent abnormality detected by all probe combinations, and the percentage detected by each was similar, 48% (X, Y, 18), 56% (2, 7, 18(, and 50% (13, 16, 18, 21, 22). Conclusions: A probe combination of five autosomes, particularly those of clinical relevance, may be more beneficial for screening embryos from patients at risk of maternal-age-related aneuploidy. However, all three probe combinations are as efficient at identifying postzygotic mosaicism, and may be used for identifying embryos with less potential of developing to term.

Normal birth following PGD for reciprocal translocation after serial vitrification of oocytes from a poor responder: a case report.

This case study reports the first successful birth outcome following preimplantation genetic diagnosis (PGD) for a chromosome translocation in embryos generated by serial vitrification of oocytes. A couple presented to the fertility clinic with 2 years of primary infertility. The woman was diagnosed with poor ovarian reserve and her partner was diagnosed with severe oligoteratozoospermia and the reciprocal translocation 46,XY,t(1;7)(p36.1;q11.23). Following counselling, the couple opted for serial vitrification of oocytes followed by PGD. A total of 31 oocytes were obtained in five egg collection cycles over a period of 12 months and 27 metaphase-II oocytes were vitrified. Nineteen of the 27 vitrified oocytes survived warming: 14 oocytes from the vitrified group and three oocytes from the fresh cycle were fertilized by intracytoplasmic sperm injection. Eleven embryos, including three from the fresh cycle, were biopsied on day 3 post insemination. Fluorescence in-situ hybridization was performed for the specific chromosomes involved in translocation. Only two embryos from the cryopreservation cycles were diagnosed as normal/balanced, one of which was transferred on day 5 post insemination. A normal healthy female infant was born at week 42 of gestation.