Identification of common genetic polymorphisms associated with down-regulated gonadotropin levels in an exome-wide association study.

OBJECTIVE: To investigate whether common genetic polymorphisms are associated with gonadotropin levels after down-regulation with daily gonadotropin-releasing hormone agonist and whether the polymorphisms of candidate variants influence the ovarian response to exogenous gonadotropins. DESIGN: Genetic association study. SETTING: University-affiliated in vitro fertilization center. PATIENTS: Subjects enrolled in an exploratory exome-wide association study (n = 862), a replication exome-wide association study (n = 86), and a classifier validation study (n = 148) were recruited from September 2016 to October 2018, September 2019 to September 2020, and January 2021 to December 2021, respectively. The included patients were aged ≤40 years and had a basal follicle-stimulating hormone (FSH) ≤12 IU/L. INTERVENTIONS: All participants received a luteal phase down-regulation long protocol. Genome DNA was extracted from the peripheral blood leukocytes. For the exploratory and replication cohorts, exome sequencing was conducted on a HiSeq 2500 sequencing platform. The multiplex polymerase chain reaction amplification technique and next-generation sequencing also were performed in the exploratory and replication cohorts. For the samples of the validation cohort, Sanger sequencing was performed. MAIN OUTCOME MEASURES: The primary endpoint was the gonadotropin levels after down-regulation, and the secondary endpoints were hormone levels and follicle diameters during stimulation, the total dose of FSH, duration of FSH stimulation, number of oocytes retrieved, and clinical pregnancy rate. RESULTS: In the exploratory cohort, we identified that FSHB rs6169 (P=2.71 × 10-24) and its single-nucleotide polymorphisms in high linkage disequilibrium were associated with the down-regulated FSH level. The same locus was confirmed in the replication cohort. Women carrying the C allele of FSHB rs6169 exhibited higher average estradiol level during stimulation (P=6.82 × 10-5), shorter duration of stimulation, and less amount of exogenous FSH (Pduration=0.0002; Pdose=0.0024). In the independent validation set, adding rs6169 genotypes into the prediction model for FSH level after down-regulation enhanced the area under the curve from 0.560 to 0.712 in a logistic regression model, and increased prediction accuracy by 41.05% when a support vector machine classifier was applied. CONCLUSION: The C allele of FSHB rs6169 is a susceptibility site for the relatively high level of FSH after down-regulation, which may be associated with increased ovarian FSH sensitivity.

The earlier the initiation of gonadotropin in poor responders in luteal phase stimulation protocols, the better.

Introduction: Luteal-phase ovarian stimulation has been proved to be feasible for producing competent oocytes/embryos and achieving live births, yet there is no standardized stimulation protocol for luteal-phase ovarian stimulation (LPS). The aim of this study was to explore the optimal timing of gonadotropin initiation in the LPS protocol for poor ovarian responders. Methods: This was a retrospective cohort study conducted in the reproductive medicine center of a tertiary hospital. A total of 327 poor responders fulfilling Bologna criteria underwent LPS with IVF/ICSI treatment. HMG and letrozole were administrated after ovulation. Patients were stratified into three groups according to the gonadotropin start day: early, early-mid, and mid-late luteal phase. A freeze-all strategy was performed for all cycles. The duration of ovarian stimulation, total gonadotropin dose, number of oocytes retrieved, implantation rate, clinical pregnancy rate, and live birth rate after frozen/thawed embryo transfer cycles were included for evaluation. Results: The group accepted ovarian stimulation in the earlier phase tended to have a shorter duration of ovarian stimulation [8 (7,10) in early luteal group, 9 (8,10.25) in early-mid luteal group, and 11 (10,12) in mid-late luteal group; P <0.001] and lower gonadotropin consumption [1993.35 ± 720.31, 2282.73 ± 703.38, and 2764.83 ± 722.26, respectively; P <0.001]. Logistic regression and multiple linear regression were used to assess the associations between the phase of gonadotropin initiation and duration of ovarian stimulation (or total gonadotropin dose) by adjusting for confounding factors. Compared with the early luteal group, longer ovarian stimulation(>9 days) was more likely to occur in the early-mid and mid-late luteal groups, with the adjusted odds ratios 0.584 (0.327-1.042) and 0.116 (0.049-0.271), respectively (P-trend<0.001). Delayed gonadotropin initiation showed an 113.200 IU increase (95%CI: 70.469, 155.930) per-day in the total gonadotropin dosage. Meanwhile, there were no significant differences in the mean number of oocytes, utilizable embryos, pregnancy outcomes among three groups. Conclusion: Although the timing of gonadotropin initiation is not associated with pregnancy outcomes, earlier initiation of gonadotropin therapy after ovulation was associated with a shorter duration of ovarian stimulation and lower gonadotropin consumption in poor responders in LPS.

Impact of Luteinized Unruptured Follicles on Clinical Outcomes of Natural Cycles for Frozen/Thawed Blastocyst Transfer.

Objective: To investigate the impact of luteinized unruptured follicles (LUF) on clinical outcomes of frozen/thawed embryo transfer (FET) of blastocysts. Methods: In this retrospective cohort study, 2,192 patients who had undergone blastocyst FET treatment with natural cycles from October 2014 to September 2017 were included. Using propensity score matching, 177 patients diagnosed with LUF (LUF group) were matched with 354 ovulating patients (ovulation group). The LUF group was further stratified by the average LH peak level of 30 IU/L. Clinical pregnancy rate and live birth rate were retrospectively analyzed between the LUF and ovulation groups, as well as between LUF subgroups. Results: After propensity score matching, general characteristics were similar in the LUF and ovulation groups. Clinical pregnancy rate in the LUF group was significantly lower than that in the ovulation group (47.46 vs. 58.76%, respectively, adjusted P = 0.01, OR 0.60, 95% CI 0.42-0.87). However, no significant difference was detected in live birth rate, although it was lower in the LUF group (43.50 vs. 50.00%, adjusted P = 0.19, OR 0.76, 95% CI 0.51-1.14). In the LUF subgroup analysis, both clinical pregnancy rate (43.02 vs. 62.30%, adjusted P = 0.02, OR 0.45, 95% CI 0.23-0.87) and live birth rate (37.21 vs. 59.02%, adjusted P = 0.01, OR 0.40, 95% CI 0.20-0.78) in the LH <30 IU/L subgroup were significantly lower than those in the LH ≥30 IU/L subgroup. Conclusion: LUF negatively affected clinical outcomes of frozen/thawed embryo transfer of blastocysts, particularly when the LH surge was inadequate.

Role of gene polymorphisms related to progesterone elevation in women undergoing long GnRH agonist protocols.

RESEARCH QUESTION: Can single-nucleotide polymorphisms (SNP) of genes related to progesterone synthesis predict the risk of premature serum progesterone elevation in women undergoing gonadotrophin-releasing hormone agonist protocols for ovarian stimulation? DESIGN: A total of 765 women were divided into high progesterone and normal progesterone groups according to progesterone concentration on the day of human chorionic gonadotrophin (HCG) administration, with the 75th percentile as the threshold between the group. Associations were analysed of genetic information from whole exome sequencing and the clinical characteristics of the two groups to identify the relationship between SNP, haplotypes and serum progesterone elevation. RESULTS: Among 40 common SNP of eight genes (FSHR, LHCGR, ESR1, ESR2, PGR, HSD3B1, CYP11A1 and CYP17A1), no statistical significance between the high and normal progesterone groups was identified in the distribution of genotypes and allele frequencies after multiple test correction to adjust the false discovery rate (PFDR > 0.05). When compared with the most common haplotypes of each gene, haplotype GAAG in CYP17A1 was associated with a 1.44-fold increased risk of progesterone elevation (95% confidence interval [CI] 1.22-1.69, PFDR < 0.001), while haplotypes of the following genes showed a decreased risk of progesterone elevation: haplotype CC in FSHR and LHCGR (0.66-fold, PFDR = 0.020, and 0.64-fold, PFDR < 0.001, respectively), CA in ESR1 (0.90-fold, PFDR < 0.001), TCTGG in ESR2 (0.92-fold, PFDR = 0.007) and GAACC in HSD3B1 (0.42-fold, PFDR < 0.001). CONCLUSIONS: Polymorphism in genes involved in enzymes or hormone receptors in the progesterone synthesis pathway may have a role in modifying risk of serum progesterone elevation.

Genotyping single-sperm cells by universal MARSALA enables the acquisition of linkage information for combined pre-implantation genetic diagnosis and genome screening.

PURPOSE: This paper aims to investigate the feasibility of performing pre-implantation genetic diagnosis (PGD) and pre-implantation genetic screening (PGS) simultaneously by a universal strategy without the requirement of genotyping relevant affected family members or lengthy preliminary work on linkage analysis. METHODS: By utilizing a universal Mutated Allele Revealed by Sequencing with Aneuploidy and Linkage Analyses (MARSALA) strategy based on low depth whole genome sequencing (~3x), not involving specific primers' design nor the enrichment of SNP markers for haplotype construction. Single-sperm cells and trephectoderm cells from in vitro fertilized embryos from a couple carrying HBB mutations were genotyped. Haplotypes of paternal alleles were constructed and investigated in embryos, and the chromosome copy number profiles were simultaneously analyzed. RESULTS: The universal MARSALA strategy allows the selection of a euploid embryo free of disease mutations for in uterus transfer and successful pregnancy. A follow-up amniocentesis was performed at 17 weeks of gestation to confirm the PGD/PGS results. CONCLUSION: We present the first successful PGD procedure based on genotyping multiple single-sperm cells to obtain SNP linkage information. Our improved PGD/PGS procedure does not require genotyping the proband or relevant family members and therefore can be applicable to a wider population of patients when conducting PGD for monogenic disorders.

Effect of the initiation of progesterone supplementation in in vitro fertilization-embryo transfer outcomes: a prospective randomized controlled trial.

OBJECTIVE: To analyze the influence of the start point of luteal support on clinical pregnancy rate, implantation rate, and live birth rate of in vitro fertilization and embryo transfer (IVF-ET) cycles. DESIGN: Single-center prospective randomized controlled trial. SETTING: University-affiliated IVF unit. PATIENT(S): Women ≤35 years of age with day 3 FSH levels <15 mIU/mL, who were undergoing their first IVF-ET cycles and received ovarian stimulation with the use of a GnRH agonist long protocol. INTERVENTION(S): The patients were randomized on the day of hCG trigger to receive luteal phase support either 1 day after oocyte retrieval (group A) or on the day of oocyte retrieval (group B). MAIN OUTCOME MEASURE(S): Clinical pregnancy rate, implantation rate, miscarriage rate in the first trimester of pregnancy, and live birth rate per embryo transfer cycle. RESULT(S): Two hundred thirty-three patients were enrolled in this study: 117 were assigned to group A and 116 to group B. The clinical pregnancy rate (group A vs. group B: 55.3% vs. 51.5%), implantation rate (38.4% vs. 38.0%), and miscarriage rate (7.7% vs. 7.5%) were similar between the two groups. The live birth rate also did not significantly differ between the two groups (45.7% vs. 46.6%). CONCLUSION(S): Our study indicated that the initiation of progesterone supplementation 1 day after oocyte retrieval did not decrease the clinical pregnancy rate, implantation rate, or live birth rate in women undergoing IVF-ET cycles with the use of the GnRH agonist long protocol. CLINICAL TRIAL REGISTRATION NUMBER: ChiCTR-IPR-14005293.

Preliminary analysis of numerical chromosome abnormalities in reciprocal and Robertsonian translocation preimplantation genetic diagnosis cases with 24-chromosomal analysis with an aCGH/SNP microarray.

PURPOSE: The aim of this study was to determine whether an interchromosomal effect (ICE) occurred in embryos obtained from reciprocal translocation (rcp) and Robertsonian translocation (RT) carriers who were following a preimplantation genetic diagnosis (PGD) with whole chromosome screening with an aCGH and SNP microarray. We also analyzed the chromosomal numerical abnormalities in embryos with aneuploidy in parental chromosomes that were not involved with a translocation and balanced in involved parental translocation chromosomes. METHODS: This retrospective study included 832 embryos obtained from rcp carriers and 382 embryos from RT carriers that were biopsied in 139 PGD cycles. The control group involved embryos obtained from age-matched patient karyotypes who were undergoing preimplantation genetic screening (PGS) with non-translocation, and 579 embryos were analyzed in the control group. A single blastomere at the cleavage stage or trophectoderm from a blastocyst was biopsied, and 24-chromosomal analysis with an aCGH/SNP microarray was conducted using the PGD/PGS protocols. Statistical analyses were implemented on the incidences of cumulative aneuploidy rates between the translocation carriers and the control group. RESULTS: Reliable results were obtained from 138 couples, among whom only one patient was a balanced rcp or RT translocation carrier, undergoing PGD testing in our center from January 2012 to June 2014. For day 3 embryos, the aneuploidy rates were 50.7% for rcp carriers and 49.1% for RT carriers, compared with the control group, with 44.8% at a maternal age < 36 years. When the maternal age was ≥ 36 years, the aneuploidy rates were increased to 61.1% for rcp carriers, 56.7% for RT carriers, and 60.3% for the control group. There were no significant differences. In day 5 embryos, the aneuploidy rates were 24.5% for rcp carriers and 34.9% for RT carriers, compared with the control group with 53.6% at a maternal age < 36 years. When the maternal age was ≥ 36 years, the aneuploidy rates were 10.7% for rcp carriers, 26.3% for RT carriers, and 57.1% for the control group. The cumulative aneuploidy rates of chromosome translocation carriers were significantly lower than the control group. No ICE was observed in cleavage and blastocyst stage embryos obtained from these carriers. Additionally, the risk of chromosomal numerical abnormalities was observed in each of the 23 pairs of autosomes or sex chromosomes from day 3 and day 5 embryos. CONCLUSION: There was not enough evidence to prove that ICE was present in embryos derived from both rcp and RT translocation carriers, regardless of the maternal age. However, chromosomal numerical abnormalities were noticed in 23 pairs of autosomes and sex chromosomes in parental structurally normal chromosomes. Thus, 24-chromosomal analysis with an aCGH/SNP microarray PGD protocol is required to decrease the risks of failure to diagnose aneuploidy in structurally normal chromosomes.

Temporal effects of human chorionic gonadotropin on expression of the circadian genes and steroidogenesis-related genes in human luteinized granulosa cells.

OBJECTIVE: It has been shown in animal models that circadian clock exists in corpora luteum which is essential for maintaining pregnancy. However, it is unknown whether circadian clock exists in corpora luteum and its relation with steroidogenesis in human ovary. STUDY DESIGN: Human luteinized granulosa cells from patients who underwent in vitro fertilization treatment were purified and cultured in vitro. Accumulation patterns of circadian gene and steroidogenesis-related gene mRNAs in human luteinized granulosa cells were observed during the 48 hours after treatment with human chorionic gonadotropin (hCG) by quantitative PCR. RESULTS: We found that the circadian genes CLOCK, PER2, and BMAL1 were expressed in cultured human luteinized granulosa cells. Among these genes, only expression of PER2 displayed oscillating patterns with a 16-h period in these cells after stimulation by hCG. Expression of CLOCK and BMAL1 did not show significant oscillating patterns. Expression of the steroidal acute regulatory protein (STAR) gene showed an oscillating pattern that was similar to that of PER2. Expression of CYP11A1, HSD3B2, and CYP19A1 increased significantly after hCG stimulation; however, none of these genes displayed significant oscillating patterns. CONCLUSIONS: Molecular circadian clock exists in human luteinized granulosa cells and may be related with steroidogenesis in human ovary.

Expression pattern of circadian genes and steroidogenesis-related genes after testosterone stimulation in the human ovary.

BACKGROUND: Previous studies have shown that circadian genes might be involved in the development of polycystic ovarian syndrome (PCOS). Hyperandrogenism is a hallmark feature of PCOS. However, the effect of hyperandrogenism on circadian gene expression in human granulosa cells is unknown, and the general expression pattern of circadian genes in the human ovary is unclear. METHODS: Expression of the circadian proteins CLOCK and PER2 in human ovaries was observed by immunohistochemistry. The mRNA expression patterns of the circadian genes CLOCK, PER2, and BMAL1, and the steroidogenesis-related genes STAR, CYP11A1, HSD3B2, and CYP19A1 in cultured human luteinized granulosa cells were analyzed over the course of 48 h after testosterone treatment by quantitative polymerase chain reaction. RESULTS: Immunostaining of CLOCK and PER2 protein was detected in the granulosa cells of dominant antral follicles but was absent in the primordial, primary, or preantral follicles of human ovaries. After testosterone stimulation, expression of PER2 showed an oscillating pattern, with two peaks occurring at the 24th and 44th hours; expression of CLOCK increased significantly to the peak at the 24th hour, whereas expression of BMAL1 did not change significantly over time in human luteinized granulosa cells. Among the four steroidogenesis-related genes evaluated, only STAR displayed an oscillating expression pattern with two peaks occurring at the 24th and 40th hours after testosterone stimulation. CONCLUSIONS: Circadian genes are expressed in the dominant antral follicles of the human ovary. Oscillating expression of the circadian gene PER2 can be induced by testosterone in human granulosa cells in vitro. Expression of STAR also displayed an oscillating pattern after testosterone stimulation. Our results indicate a potential relationship between the circadian clock and steroidogenesis in the human ovary, and demonstrate the effect of testosterone on circadian gene expression in granulosa cells.

Influence of menstrual cycle on the expression of clock genes in peripheral blood mononuclear cells in Macaca fascicularis.

OBJECTIVE: To investigate whether the expression patterns of periphery clock genes were influenced by menstrual cycle in a monkey model. STUDY DESIGN: In this preliminary study, the expression patterns of four clock genes (Bmal1, Clock, Cry1 and Per2) in peripheral blood mononuclear cells (PBMCs) from 6 female Macaca fascicularis in menstrual, late follicular and mid luteal phases of menstrual cycle were determined by qrt-PCR. RESULTS: Bmal1 and Per2 mRNA levels were found to exhibit significant diurnal rhythms in all phases of the menstrual cycle. The expression of Cry1 mRNA was statistically rhythmic in late follicular and mid luteal phases. A main effect of menstrual cycle existed on the rhythms of Bmal1, Cry1 and Per2 expression, but not Clock expression. No significant differences were detected between menstrual phase and late follicular phase in all clock genes. Significant differences were found on the expression of Bmal1, Cry1 or Per2 mRNA between late follicular phase and mid luteal phase, when no difference existed in estrogen level, indicating the role of progesterone on biological clock gene expression. Furthermore, the peak of Bmal1 mRNA level slightly advanced in mid luteal phase compared with that in menstrual and late follicular phases. CONCLUSION: The expression patterns of clock genes in PBMCs were influenced by menstrual cycle, potentially by the change of progesterone levels, and this effect maybe correlated with early pregnancy.

Self-control study on reduced-dose depot versus daily administration of gonadotrophin-releasing hormone agonists for pituitary desensitization in in vitro fertilization cycles.

AIM: Our aim was to analyze the effect of reducing the dose of depot gonadotrophin-regulating hormone-agonist (GnRH-a) to 1.0 mg on pituitary desensitization and clinical outcome of in vitro fertilization and embryo transfer cycles. MATERIAL AND METHODS: This retrospective self-control study was conducted on 143 patients who underwent repeated long-protocol treatment from 1 January 2011 to 31 May 2012 at our hospital. Of the 143 patients, 64 received reduced-dose depot (1.0 mg diphereline depot) GnRH-a for the first cycle and short-acting GnRH-a (0.05 mg diphereline) for the second cycle, while 79 patients received short-acting GnRH-a for the first cycle and reduced-dose depot GnRH-a for the second cycle. RESULTS: The serum follicle-stimulating hormone, luteinizing hormone and estradiol levels on the day of gonadotrophin initiation were significantly higher in the short-acting group compared with the long-acting group. Both number of days of gonadotrophin stimulation and gonadotrophin doses were significantly higher in the short-acting group. On the day of human chorionic gonadotrophin administration, the serum estradiol level was significantly higher while the progesterone level was significantly lower in the short-acting group. There were no significant differences with regard to the number of retrieved oocytes, fertilization rate, number of transferred embryos, clinical pregnancy rate, implantation rate, and early pregnancy loss rate between the two groups. However, the oocyte maturation rate was significantly higher in the long-acting group. CONCLUSION: Reduced-dose depot GnRH-a can be successfully used for pituitary desensitization in in vitro fertilization and embryo transfer. Deeper downregulation with reduced-dose depot GnRH-a indicates that the optimal dose of GnRH-a warrants future study.

[Clinical investigation to compare aCGH and FISH in preimplantationgenetic diagnosis of chromosome translocation carriers].

OBJECTIVE: To investigate the clinical use of array comparative genomic hybridization (aCGH) with fluorescence in situ hybridization (FISH) in preimplantion genetic diagnosis (PGD) for reciprocal and Robertsonian translocation carriers. METHODS: From Jan. 2012 to Jun. 2013, a total of 220 PGD cycles from 151 reciprocal translocation and 62 Robertsonian translocation carrier couples, including 33 cycles for reciprocal translocation carriers and 22 cycles for Robertsonian translocation carriers performed using array CGH, and 119 cycles for reciprocal translocation carriers and 46 cycles for Robertsonian translocation carriers performed using FISH were retrospectively studied. The rate of accurate diagnosis was compared between two methods. RESULTS: Normal and/or balance rates of the two translocated chromosomes detected by aCGH for both reciprocal and Robertsonian translocation carriers were 38.20% (123/322) and 67.20% (127/189), significantly higher than 15.39% (195/1 267) and 30.75% (202/657) by FISH (all P < 0.05). Abnormal rates of the two translocated chromosomes detected by aCGH for both reciprocal and Robertsonian translocation carriers were 59.32% (191/322) and 30.69% (58/189), significantly lower than 83.03% (1 052/1 267) and 67.43% (443/657) by FISH (all P < 0.05). And the rate of aneu ploidy in non-translocated chromosome from reciprocal translocation embryos was 20.19% (65/322), which was significantly lower than 38.62% (13/189) from Robertsonian translocation embryos (P < 0.01). CONCLUSIONS: Normal and/or balance rates of the two translocated chromosomes detected by array CGH were significantly higher than FISH. And the rate of aneuploidy in non-translocated chromosomes from reciprocal translocation embryos was significantly lower than that from Robertsonian translocation embryos.

Preliminary investigation of the impact of anticentromere antibody on oocyte maturation and embryo cleavage.

OBJECTIVE: To explore whether anticentromere antibody (ACA) is the most significant antibody among antinuclear antibodies (ANA), which adversely affect oocyte maturation, embryo cleavage, and pregnancy outcome in women undergoing an intracytoplasmic sperm injection program. DESIGN: Retrospective, nested case-control study. SETTING: Center for reproductive medicine, university hospital. PATIENT(S): A total of 187 women receiving the first intracytoplasmic sperm injection cycle were enrolled in this study, including 20 women with positive ACA and ANA (ACA[+]/ANA[+] group), 51 women with negative ACA and positive ANA(ACA[-]/ANA[+] group), and 116 patients with negative ACA and ANA (ACA[-]/ANA[-] group). Patients in the three groups were age-matched. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Percentages of germinal vesicle, metaphase I, and metaphase II oocytes, embryo cleavage rate, number of high-quality embryos, and rates of pregnancy and implantation. RESULT(S): The metaphase I oocyte percentage was markedly higher and the metaphase II oocyte percentage and the normal cleavage rate were significantly lower in the ACA[+]/ANA[+] group as compared with the ACA[-]/ANA[+] group. Furthermore, statistically significant differences were found in rates of pregnancy and implantation among the three groups. However, no significant difference was found between any two groups owing to the small sample size, except for a significantly lower implantation rate being found in the ACA[+]/ANA[+] group when compared with the ACA[-]/ANA[-] group. CONCLUSION(S): Our data suggest that ACA may be the essential marker for defective oocytes or embryos in infertile women with any type of ANA.

[A preliminary study on the application of array comparative genomic hybridization for preimplantaion genetic diagnosis].

OBJECTIVE: To assess the value of array comparative genomic hybridization (array CGH) technique for preimplantation genetic diagnosis (PGD). METHODS: Array CGH was performed on three types of cells, which included 3-5 cells isolated from B2/C38/A1 embryonic stem cell lines, single cells isolated from two discarded normal fertilized embryos, and 10 blastocysts biopsied from 5 couples undergoing PGD for chromosomal translocations. For the 10 blastocysts, 8 were abnormal embryos, 1 appeared to be normal but showed arrested development, and 1 embryo was without any fluorescence signals. 24sure V3 or 24sure+ array chips were applied for CGH analysis. The results were analyzed with a BlueFuse Multi software. RESULTS: (1) The results of cells from B2/C3/A1 embryo stem cells by array CGH were consistent with karyotyping analysis. (2) For the 6 single cell samples from two discarded embryos, 2 blastomeres from one embryo were diagnosed as with aneuploidy and a normal karyotype, respectively. Two out of 4 blastomeres biopsied from another embryo were normal, whilst the remaining two were diagnosed with aneuploidies of -22 and +13. Repeated detection with 24sure+ array was consistent with the 24sure V3 result. (3) Ten cell masses from 10 embryos in PGD cycles were successfully analyzed with array CGH, among which four were confirmed with fluorescence in situ hybridization (FISH) on day 3. In two of them, array CGH confirmed FISH diagnosis. For the remaining two, additional aneuploidies for chromosomes not tested by FISH were discovered by array CGH. Another embryo diagnosed as no signal by FISH was found to have trisomy 13 by array CGH. The remaining 5 embryos also showed discordant results by FISH and array CGH. One embryo from a Robertsonian translocation carrier was found to have monosomy 13 by FISH but trisomy 14 and additional aneuploidies by both 24sure V3 and 24sure+ chips. One embryo with many fragments and arrested development by D5 showed discordant results by FISH and array CGH. However, the FISH and array CGH results for other two embryos from this reciprocal translocation carrier were consistent. Three embryos with inconsistent results by FISH and array CGH had a chromosomal translocation involving q11 region. CONCLUSION: Array CGH is useful for PGD applications for its capability to detect structural chromosomal abnormalities through screening of aneuploidies. However, the 24sure V3 array may not suit detection of translocations with breakpoints close to the q11 region of chromosomes.

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

OBJECTIVE: To determine whether a new assisted hatching (AH) method increases the implantation and clinical pregnancy rates of frozen-thawed day-3 (D3) embryos. DESIGN: Prospective study. SETTING: A university hospital in vitro fertilization (IVF) program. PATIENT(S): Patients 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. INTERVENTION(S): The 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. MAIN OUTCOME MEASURE(S): Clinical pregnancy and implantation rates. RESULT(S): The 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. CONCLUSION(S): Mechanically expanding the ZP of frozen-thawed D3 embryos with injected hydrostatic pressure after thawing increases the implantation rate compared with control embryos.

A randomised comparison of side effects and patient inconvenience of two vaginal progesterone formulations used for luteal support in in vitro fertilisation cycles.

OBJECTIVE: To compare side effects and patient inconvenience of two vaginal progesterone (P) formulations for luteal support in in vitro fertilisation cycles. STUDY DESIGN: Sixty infertile patients at risk of developing ovarian hyperstimulation syndrome were randomised to receive either Cyclogest vaginal suppositories 400mg twice daily or Crinone 8% vaginal gel once daily for 14 days as the luteal support. On Day 6 and Day 16 after embryo transfer, they rated side effects and patient inconvenience into four grades: none, mild, moderate and severe by completing a questionnaire. RESULTS: Perineal irritation was reported by about 20% of patients in each group. Significantly more patients using Cyclogest suppositories graded inconvenience of administration, leaking out and interference with coitus as moderate or severe. CONCLUSION: There was no difference in perineal irritation after Cyclogest suppositories or Crinone 8% gel although significantly more patients found inconvenience of administration, leaking out and interference with coitus after Cyclogest.

A randomized double-blind study to compare the effectiveness of three different doses of lignocaine used in paracervical block during oocyte retrieval.

PURPOSE: To compare the effectiveness of three different doses of lignocaine used in paracervical block (PCB) during transvaginal ultrasound-guided oocyte retrieval (TUGOR) METHODS: In this double-blind study, 153 patients undergoing TUGOR in their first in vitro fertilization cycle were randomized to receive 50, 100, and 150 mg of lignocaine in PCB. Pain levels were measured by a 100-mm linear visual analogue scale (0 = none to 100 = intolerable). RESULTS: No differences were seen in the demographic data, the ovarian responses, the duration of TUGOR, and the number of follicles punctured. Vaginal and abdominal pain levels during TUGOR and 4 h after TUGOR were not significantly different among the three groups The median vaginal and abdominal pain levels during the retrieval were 22.0-24.0 and 30.0-32.0 respectively. CONCLUSION: The use of 50 mg of lignocaine is recommended in PCB because of the lack of improvement in pain relief on higher doses and potential dose-related risks.

Anxiolytic premedication reduces preoperative anxiety and pain during oocyte retrieval. A randomized double-blinded placebo-controlled trial.

BACKGROUND: The role of anxiolytic premedication remains unclear and significant postoperative side-effects may result from routine use. METHODS: In this double-blinded study, 100 infertile patients were randomized on the day of ultrasound-guided oocyte retrieval (TUGOR) by a computer-generated randomization list in sealed envelopes to receive either (i) 50 mg pethidine and 25 mg promethazine (premedication group) or (ii) normal saline (placebo group) i.m. 30 min prior to TUGOR. Anxiety level, pain levels and severity of postoperative side-effects were recorded. RESULTS: No differences were seen in demographic data, TUGOR duration, number of follicles punctured and clinical outcome. Preoperative anxiety level was significantly higher than the basal anxiety level in the placebo group only. The vaginal and abdominal pain levels during TUGOR and 4 h after TUGOR were significantly higher in the placebo group than the premedication group. Significantly more patients complained of drowsiness after TUGOR in the premedication group than the placebo group and other side-effects were comparable in both groups. CONCLUSION: Routine use of anxiolytic premedication prevented an increase of preoperative anxiety level, reduced pain levels during oocyte retrieval but was associated with a higher percentage of moderate/severe drowsiness in the postoperative period.