Human oocytes harboring damaged DNA can complete meiosis I.

OBJECTIVE: To determine whether human oocytes possess a checkpoint to prevent completion of meiosis I when DNA is damaged. DESIGN: DNA damage is considered a major threat to the establishment of healthy eggs and embryos. Recent studies found that mouse oocytes with damaged DNA can resume meiosis and undergo germinal vesicle breakdown (GVBD), but then arrest in metaphase of meiosis I in a process involving spindle assembly checkpoint (SAC) signaling. Such a mechanism could help prevent the generation of metaphase II (MII) eggs with damaged DNA. Here, we compared the impact of DNA-damaging agents with nondamaged control samples in mouse and human oocytes. SETTING: University-affiliated clinic and research center. PATIENT(S): Patients undergoing ICSI cycles donated GV-stage oocytes after informed consent; 149 human oocytes were collected over 2 years (from 50 patients aged 27-44 years). INTERVENTIONS(S): Mice and human oocytes were treated with DNA-damaging drugs. MAIN OUTCOME MEASURE(S): Oocytes were monitored to evaluate GVBD and polar body extrusion (PBE), in addition to DNA damage assessment with the use of γH2AX antibodies and confocal microscopy. RESULT(S): Whereas DNA damage in mouse oocytes delays or prevents oocyte maturation, most human oocytes harboring experimentally induced DNA damage progress through meiosis I and subsequently form an MII egg, revealing the absence of a DNA damage-induced SAC response. Analysis of the resulting MII eggs revealed damaged DNA and chaotic spindle apparatus, despite the oocyte appearing morphologically normal. CONCLUSION(S): Our data indicate that experimentally induced DNA damage does not prevent PBE in human oocytes and can persist in morphologically normal looking MII eggs.

No. 354-Canadian HIV Pregnancy Planning Guidelines.

OBJECTIVE: The objective of the Canadian HIV Pregnancy Planning Guidelines is to provide clinical information and recommendations for health care providers to assist Canadians affected by HIV with their fertility, preconception, and pregnancy planning decisions. These guidelines are evidence- and community-based and flexible and take into account diverse and intersecting local/population needs based on the social determinants of health. INTENDED OUTCOMES: EVIDENCE: Literature searches were conducted by a librarian using the Medline, Cochrane Central Register of Controlled Trials (CENTRAL), and Embase databases for published articles in English and French related to HIV and pregnancy and HIV and pregnancy planning for each section of the guidelines. The full search strategy is available upon request. VALUES: The evidence obtained was reviewed and evaluated by the Infectious Diseases Committee of the SOGC under the leadership of the principal authors, and recommendations were made according to the guidelines developed by the Canadian Task Force on Preventive Health Care and through use of the Appraisal of Guidelines Research and Evaluation instrument for the development of clinical guidelines. BENEFITS, HARMS, AND COSTS: Guideline implementation should assist the practitioner in developing an evidence-based approach for the prevention of unplanned pregnancy, preconception, fertility, and pregnancy planning counselling in the context of HIV infection. VALIDATION: These guidelines have been reviewed and approved by the Infectious Disease Committee and the Executive and Council of the SOGC. SPONSOR: Canadian Institutes of Health Research Grant Planning and Dissemination grant (Funding Reference # 137186), which funded a Development Team meeting in 2016.

Tri-directional anaphases as a novel chromosome segregation defect in human oocytes.

STUDY QUESTION: What are the chromosome segregation errors in human oocyte meiosis-I that may underlie oocyte aneuploidy? SUMMARY ANSWER: Multiple modes of chromosome segregation error were observed, including tri-directional anaphases, which we attribute to loss of bipolar spindle structure at anaphase-I. WHAT IS KNOWN ALREADY: Oocyte aneuploidy is common and associated with infertility, but mechanistic information on the chromosome segregation errors underlying these defects is scarce. Lagging chromosomes were recently reported as a possible mechanism by which segregation errors occur. STUDY DESIGN, SIZE, DURATION: Long-term confocal imaging of chromosome dynamics in 50 human oocytes collected between January 2015 and May 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: Germinal vesicle (GV) stage oocytes were collected from women undergoing intracytoplasmic sperm injection cycles and also CD1 mice. Oocytes were microinjected with complementary RNAs to label chromosomes, and in a subset of oocytes, the meiotic spindle. Oocytes were imaged live through meiosis-I using confocal microscopy. 3D image reconstruction was used to classify chromosome segregation phenotypes at anaphase-I. Segregation phenotypes were related to spindle dynamics and cell cycle timings. MAIN RESULTS AND THE ROLE OF CHANCE: Most (87%) mouse oocytes segregated chromosomes with no obvious defects. We found that 20% of human oocytes segregated chromosomes bi-directionally with no lagging chromosomes. The rest were categorised as bi-directional anaphase with lagging chromosomes (20%), bi-directional anaphase with chromatin mass separation (34%) or tri-directional anaphase (26%). Segregation errors correlated with chromosome misalignment prior to anaphase. Spindles were tripolar when tri-directional anaphases occurred. Anaphase phenotypes did not correlate with meiosis-I duration (P = 0.73). LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Oocytes were recovered at GV stage after gonadotrophin-stimulation, and the usual oocyte quality caveats apply. Whilst the possibility that imaging may affect oocyte physiology cannot be formally excluded, detailed controls and justifications are presented. WIDER IMPLICATIONS OF THE FINDINGS: This is one of the first reports of live imaging of chromosome dynamics in human oocytes, introducing tri-directional anaphases as a novel potential mechanism for oocyte aneuploidy. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by grants from Fondation Jean-Louis Lévesque (Canada), CIHR (MOP142334) and CFI (32711) to GF. JH is supported by Postdoctoral Fellowships from The Lalor Foundation and CIHR (146703). The authors have no conflict of interest.

450 IU versus 600 IU gonadotropin for controlled ovarian stimulation in poor responders: a randomized controlled trial.

OBJECTIVE: To compare the outcomes of controlled ovarian stimulation/in vitro fertilization cycles using 450 IU and 600 IU gonadotropin per day in women at risk of poor ovarian response. DESIGN: Prospective randomized controlled nonblinded study. SETTING: University-affiliated private IVF center. PATIENT(S): Women considered to be at risk of poor ovarian response: aged <41 years with basal FSH >10 IU/L, antimüllerian hormone <1 ng/mL, antral follicle count ≤ 8, or a previous IVF cycle with ≥ 300 IU/d gonadotropin that resulted in a cancellation, <8 follicles, or <5 oocytes. INTERVENTION(S): A total of 356 patients underwent a microdose GnRH agonist flare-up IVF/intracytoplasmic sperm injection protocol with a fixed daily dose of either 450 IU FSH (n = 176) or 600 IU FSH (n = 180) equally divided between Menopur and Bravelle. MAIN OUTCOME MEASURE(S): Number of mature oocytes retrieved. RESULT(S): The two groups were similar in terms of age, ovarian reserve, cause of infertility, duration of stimulation, and cycle cancellation rate. There were no significant differences in the number of metaphase II oocytes retrieved (4 [range 0-6] vs. 4 [range 2-7]), fertilization rate (62.4% vs. 57.0%), biochemical pregnancy rate (20.5% vs. 22.9%), clinical pregnancy rate (16.4% vs. 18.3%), and implantation rate (29.8% vs. 30.4%) between the 450 IU and 600 IU groups, respectively. CONCLUSION(S): Gonadotropin of 600 IU/d does not improve outcome of IVF cycles compared with 450 IU/d in women at risk of poor ovarian response. CLINICAL TRIAL REGISTRATION NUMBER: NCT00971152.

An algorithm combining ultrasound monitoring and urinary luteinizing hormone testing: a novel approach for intrauterine insemination timing.

OBJECTIVE: Intrauterine insemination (IUI) is a commonly used treatment for infertility. Optimal timing of insemination is achieved either by ultrasound monitoring of follicular growth followed by the administration of human chorionic gonadotropin (hCG) or by the detection of a luteinizing hormone (LH) surge through urinary LH testing (uLH). However, in cycles where follicular growth is monitored, there is a possibility of a premature LH rise which may affect the outcome of treatment. The objective of the current study was to determine the frequency of spontaneous LH surges in ultrasound-monitored IUI cycles. METHODS: One hundred IUI cycles were followed for this prospective cohort study. In combination with ultrasound monitoring, uLH testing was performed twice daily. A serum LH test was performed in the case of an inconclusive uLH test result. IUI was performed either on the day after a positive LH test or, if the diameter of the dominant follicle reached 18 mm and the LH test was still negative, 36 hours after ovulation triggering by administration of hCG. RESULTS: Of the 87 analyzed cycles, 19 (21.8%) exhibited a premature LH surge as detected by urine testing. Eleven further cycles had an inconclusive urine result, and in six of these (6.9% of cycles) the result was confirmed positive by serum LH testing, giving a total of 25 cycles (28.7%) experiencing a premature LH surge. CONCLUSION: A considerable proportion of patients undergoing ultrasound-monitored IUI cycle had a spontaneous LH surge before ovulation triggering was scheduled. This could affect pregnancy rates following IUI.

Pregnancy loss in pregnancies conceived after in vitro oocyte maturation, conventional in vitro fertilization, and intracytoplasmic sperm injection.

OBJECTIVE: To compare rates of pregnancy loss after oocyte maturation in vitro (IVM), after IVF, and after intracytoplasmic sperm injection (ICSI). DESIGN: Retrospective comparative study. SETTING: University tertiary-care center for infertility. PATIENT(S): Women undergoing assisted reproductive technology in a single center. INTERVENTION(S): Oocyte maturation in vitro, IVF, or ICSI, as indicated. MAIN OUTCOME MEASURE(S): Biochemical pregnancy, clinical miscarriage, ectopic pregnancy, and late fetal loss. RESULT(S): There were 1,581 positive pregnancy tests (120 IVM, 849 IVF, and 612 ICSI). The biochemical pregnancy loss rate did not statistically significantly differ among the groups: 17.5% (21/120) after IVM, 17.0% (144/849) after IVF, and 18.0% (110/612) after ICSI. The clinical miscarriage rate after IVM was 25.3% (25/99), which was statistically significantly different compared with 15.7% (111/705) after IVF and 12.6% (63/502) after ICSI. However, the clinical miscarriage rates in women with polycystic ovary syndrome were statistically similar, at 24.5% (24/98) after IVM and 22.2% (18/81) after IVF. The ectopic pregnancy rates also were statistically similar: 1.0% (1/99) after IVM, 2.3% (16/705) after IVF, and 1.8% (9/502) after ICSI. The late fetal loss rates were similar as well: 1.0% (1/99) after IVM, 2.7% (19/705) after IVF, and 2.9% (14/502) after ICSI. There were no chromosomal abnormalities in the IVM group. CONCLUSION(S): There is a higher rate of clinical miscarriage after IVM when compared with IVF and ICSI. This appears to be related to polycystic ovary syndrome rather than to the IVM procedure.

Instability in the transmission of the myotonic dystrophy CTG repeat in human oocytes and preimplantation embryos.

OBJECTIVE: To elucidate the timing and variability of CTG repeat expansion within the human dystrophia myotonica protein kinase (DMPK) gene in early development. DESIGN: Triplet-primed polymerase chain reaction was used to amplify the expanded CTG repeat in oocytes and embryos obtained from myotonic dystrophy 1 (DM1) patients, and a heminested polymerase chain reaction approach was used to amplify the normal CTG repeats in supernumerary IVF embryos. SETTING: University hospital laboratory. PATIENT(S): Two DM1-affected females undergoing preimplantation genetic diagnosis who carried different CTG repeats. Also, 61 IVF patients who carried a (CTG)(5-18) and (CTG)(19-37) normal DMPK repeat. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The degree of expansion of the repeat in the oocytes and embryos compared with the DM1-affected maternal repeat and the size of the (CTG)(19-37) repeat compared with the parental size in IVF embryos. RESULT(S): The degree of repeat expansion was greater than the DM1 maternal lymphocyte for two of four oocytes, including a germinal vesicle-stage oocyte and 17 of 20 three-cell to blastocyst stage embryos. A change in the (CTG)(19-37) repeat was seen in 7 (7%) of 95 paternal transmissions but in no maternal transmissions. CONCLUSION(S): Because the repeat was already expanded in the immature oocyte, the initial expansion most likely occurs during oogenesis. A variable degree of DMPK(CTG)(n) expansion in the embryo is seen from different mothers. In addition, instability in paternal transmission of normal-range (CTG)(19-37) repeats occurs at the level of the embryo.

Transmission ratio distortion in the myotonic dystrophy locus in human preimplantation embryos.

One form of myotonic dystrophy, dystrophia myotonica 1 (DM1), is caused by the expansion of a (CTG)(n) repeat within the dystrophia myotonica-protein kinase (DMPK) gene located in chromosome region 19q13.3. Unaffected individuals carry alleles with repeat size (CTG)(5-37), premutation carriers (CTG)(38-49) and DM1 affected individuals (CTG)(50-6,000). Preferential transmission both of expanded repeats from DM1-affected parents and larger DMPK alleles in the normal-size range have been reported in live-born offspring. To determine the moment in development when transmission ratio distortion (TRD) for larger normal-size DMPK alleles is generated, the transmission from heterozygous parents with one repeat within the (CTG)(5-18) range (Group I repeat) and the other within the (CTG)(19-37) range (Group II repeat) to human preimplantation embryos was analysed. A statistically significant TRD of 59% (95% confidence interval of 54-64) in favour of Group II repeats from both mothers and fathers was observed in preimplantation embryos, which remained significant when female embryos were considered separately. In contrast, no significant TRD was detected for repeats from informative Group I/Group I parents. Our analysis showed that Group II repeats specifically were preferentially transmitted in human preimplantation embryos. We suggest that TRD, in Group II repeats at the DMPK locus, is likely to result from events occurring at or around the time of fertilisation.

Prospect of preimplantation genetic diagnosis for heritable mitochondrial DNA diseases.

To perform preimplantation genetic diagnosis for women carrying heteroplasmic mitochondrial DNA (mtDNA) mutations, it is necessary to ensure that the proportion of mutant mtDNA diagnosed in the biopsied cell gives an accurate indication of the mutant load in the remaining embryo. A heteroplasmic mouse model, carrying NZB and BALB mtDNA genotypes, was used to study the relative proportions of each mtDNA genotype in the ooplasm and first polar body of mature oocytes, and between blastomeres of early cleavage stage embryos. The levels of heteroplasmy varied widely in the gametes compared with the maternal genotype. However, the distribution of the two mtDNA genotypes was virtually identical between the ooplasm and polar body of a mature oocyte, and also between the blastomeres of each 2-, 4- and 6-8-cell embryo. Therefore, the level of heteroplasmy diagnosed from the polar body of an unfertilized oocyte or from a single blastomere of an embryo is representative of the level in the embryo as a whole. Reliable results were obtained from both polar bodies and blastomeres, but the efficiency of diagnosis was greater with blastomeres. We conclude that preimplantation genetic diagnosis is feasible for mtDNA diseases, although it should be approached with caution, as it is possible that transmission of some pathogenic mutations could behave in a different manner.

Consecutive transfer of day 3 embryos and of day 5-6 blastocysts increases overall pregnancy rates associated with blastocyst culture.

PURPOSE: To investigate whether the consecutive embryo transfer of day 3 embryos and of blastocyst protects against failure to reach embryo transfer and provides additional pregnancies. METHODS: An embryo transfer was performed on day 3 following which all remaining embryos were cultured to the blastocyst stage for a possible second transfer. RESULTS: One hundred and forty-two patients were selected for extended culture. Thirty-two of these patients did not develop blastocysts in culture, however, there were 12 pregnancies achieved in this group. CONCLUSIONS: The consecutive transfer of day 3 embryos and blastocysts can prevent the total loss of a cycle when embryos fail to develop to the blastocyst stage in culture and thereby provide additional pregnancies.

Genetic prenatal and preimplantation diagnosis of trinucleotide repeat disorders.

This review examines the history, present status and future of genetic antenatal diagnosis for the trinucleotide repeat disorders, including Huntington's disease, Fragile X syndrome and myotonic dystrophy. Conventional prenatal diagnosis and the relatively new field of preimplantation genetic diagnosis, which can diagnose an affected embryo before a pregnancy is established, are described. Genetic diagnosis for these late onset diseases has inherent difficulties and many controversies. However, antenatal diagnosis is an important service for an individual with one of these mutations, who wishes to prevent the birth of an affected child.