Polar body diagnosis of common aneuploidies by FISH

Purpose: The purpose of this work was to investigate the reliability and accuracy of polar body analysis for preimplantation diagnosis of common aneuploidies in IVF patients of advanced maternal age. Design: We have previously introduced polar body analysis as an approach for nondestractive evaluation of the genotype of human oocytes. The method has recently been applied in a clinical trial involving 45 infertile patients, demonstrating the feasibility of preconception diagnosis of common aneuploidies by fluorescent in situ hybridization (FISH). The present paper describes the experience of polar body diagnosis in 135 IVF patients (161 cycles) of advanced maternal age. Results: FISH results of the first and/or second polar bodies were available in 648 (72.4%) of 895 biopsied oocytes subjected to FISH analysis. Of 648 oocytes with FISH results, 208 demonstrated chromosomal abnormalities. Of 440 oocytes predicted to be free from monosomy or trisomy of chromosomes X, 18, and/or 13/21, 314 were normally fertilized, cleaved, and transferred in 122 treatment cycles, resulting in 6 healthy deliveries and 12 ongoing pregnancies following confirmation of the polar body diagnosis by CVS or amniocentesis. Conclusion: The method may be useful for detection of oocytes with common chromosomal trisomies in IVF patients of advanced maternal age.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

Prevention of Age-Related Aneuploidies by Polar Body Testing of Oocytes

Purpose: We previously demonstrated that aneuploidy-free oocytes may be preselected by testing the first and second polar bodies removed from oocytes following their maturation and fertilization. The present paper describes the results of the application of the method in 659 in vitro fertilization cycles from patients of advanced maternal age. Methods: Using micromanipulation techniques, 3943 oocytes were tested by polar body sampling and fluorescent in situ hybridization analysis using specific probes for chromosomes 13, 18, and 21. Results: Fluorescent in situ hybridization results were available for 3217 (81.6%) of 3943 oocytes studied, of which 1388 (43.1%) had aneuploidies; 35.7% of the aneuploidies were of first meiotic division origin, and 26.1% of second meiotic division origin. Most errors in the first meiotic division were represented by chromatid malsegregation. The transfer of embryos deriving from 1558 of 1829 aneuploidy-free oocytes in 614 treatment cycles resulted in 131 clinical pregnancies and 88 healthy children born after confirmation of the polar body diagnosis. Conclusions: Polar body testing of oocytes provides an accurate and reliable approach for prevention of age-related aneuploidies in in vitro fertilization patients of advanced maternal age.     

Three births after preimplantation genetic diagnosis for cystic fibrosis with sequential first and second polar body analysis

OBJECTIVE: The purpose of this study was to determine the accuracy and feasibility of sequential polar body removal and analysis for preimplantation genetic diagnosis of mendelian disorders. STUDY DESIGN: Three couples with risk factors for cystic fibrosis had preimplantation genetic diagnosis with the use of sequential polar body analysis. After stimulation, oocytes were harvested and the first polar bodies were removed and analyzed on the day of aspiration. The following day, after fertilization, the second polar bodies were aspirated. Only embryos known to have inherited the normal maternal allele were transferred. RESULTS: All three couples had successful pregnancies resulting in the births of unaffected infants. CONCLUSIONS: Preimplantation diagnosis with the use of sequential polar body removal is feasible and can prevent the establishment of genetically abnormal pregnancies for couples at risk. (Am J Obstet Gynecol 1998;178:1298-306.)     

Outcome of laser-assisted polar body biopsy and aneuploidy testing

Polar body biopsy and subsequent fluorescence in-situ hybridization (FISH) analysis allows detection of maternally derived chromosomal aneuploidies in human oocytes during IVF treatment. The development of a diode laser technique for the partial opening of the zona pellucida has stimulated the use of this technique to assist polar body biopsy. Laser-assisted polar body biopsy was performed in 140 IVF cycles from patients of advanced maternal age (> or =35 years). A total of 921 oocytes were treated by a laser for partial zona opening and polar body removal. FISH was performed for chromosomes 13, 16, 18, 21 and 22 and results were available for 903 oocytes (98%). In all, 443 oocytes (49.1%) were euploid and of these, 293 were fertilized. A total of 214 embryos were transferred in 120 embryo transfer cycles (1.78 per embryo transfer) resulting in 27 clinical pregnancies (22.5% per embryo transfer) with an implantation rate of 15.4%. Subsequently, five women aborted (18.5%) and 24 healthy children were born from the remaining 22 pregnancies, which gives a take home baby rate of 18.3% per transfer cycle. It is concluded that polar body biopsy using a diode laser system is as efficient as standard polar body biopsy using zona drilling.     

采用第一极体进行植入前诊断的实验研究

目的 :建立采用第一极体植入前染色体非整倍体诊断的方法。方法 :取试管婴儿后的未受精卵细胞 ,激光打孔法行第一极体活检 ,固定后行多色荧光原位杂交 (FISH) ,分析极体中 13,16 ,18,2 1和 2 2号染色体的核型情况。结果 :活检成功率为 94 .7% ,FISH成功率为 86 .7% ,6 1.5 %的极体核型正常 ,38.5 %的极体为非整倍体。结论 :激光打孔结合FISH法是一种快速有效的极体植入前诊断方法     

Use of PRINS for preconception screening of polar bodies for common aneuploidies

It has been shown that preconceptional screening for oocyte aneuploidies could help increase the pregnancy rate after in vitro fertilization (IVF), particularly in cases of advanced maternal age. The FISH (fluorescent in situ hybridization) technique is usually used to examine the first polar body (I-PB) for oocyte screening and so avoid fertilizing and transferring embryos from aneuploid oocytes. We have tested the feasibility of using another technique, the primed in situ (PRINS) reaction for this purpose. PRINS is a rapid, inexpensive method of labelling chromosomes. Chromosomes were labelled by in situ annealing with chromosome-specific oligonucleotide primers, followed by primer extension with labelled nucleotides using Taq DNA polymerase. A total of 183 PRINS reactions were performed with primers for chromosomes 13, 16, 18, 21 or X on 63 I-PBs removed from oocytes that failed to become fertilized during IVF. Each I-PB underwent three successive double-labelling reactions and intense signals were obtained in less than 40 min. Our data suggest that PRINS may be a useful alternative or a complement to FISH for detecting the main aneuploidies in all oocytes obtained after follicular puncture.     

FISH Analysis of Six Chromosomes in Unfertilized Human Oocytes After Polar Body Removal

Purpose: To develop an improved technique for estimatingchromosomal abnormalities in human oocytes byfluorescence in situ hybridization (FISH) and to correlate theposition of single chromatids with the chromosomal status ofthe oocytes. Methods: Oocytes that were at metaphase II about17–20 hr after insemination or intracytoplasmic sperm injection(ICSI) were treated with pronase to remove the zonapellucida and polar body (PB) and then spread on slides usingHCl and Tween 20. Two rounds of FISH were performedusing direct-labeled probes: chromosomes 1, 13, 21 (round1); chromosomes X, 7, 18 (round 2). Results: Of the 63 oocytes from 18 patients (mean age,32 years), 48 (76%) had one DNA complement as expected, 9(14%) had 2 DNA complements, 3 (5%) gave incomplete FISHsignals, and 3 (5%) were not analyzable. Of the 48 oocyteswith one set of DNA, 48% were haploid, 44% were aneuploidfor one or more chromosomes, and 8% were polyploid. Wealso found an increased frequency of predivision of chromatidbivalents in aneuploid oocytes, especially for chromosome 21. Conclusions: This technique enables simultaneousassessment of six chromosomes in human oocytes, and thereforecan be useful for accurately determining the incidence andcauses of genetic imbalances in human oocytes andapparently low fertilization rates.     

Chromosomal abnormalities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies

Most chromosomal abnormalities originate from female meiosis and contribute significantly to pregnancy failures, particularly in women of advanced maternal age. A total of 8,382 oocytes were obtained in 1,297 IVF cycles from patients of advanced maternal age (mean 38.5 years). Following a standard IVF protocol, oocytes were tested following removal and fluorescence in-situ hybridization (FISH) analysis of the first (PB1) and second polar bodies (PB2), using probes specific for chromosomes 13, 16, 18, 21 and 22 (Vysis). FISH results were available in 67,33 (80.3%) oocytes tested, 3,509 (52.1%) of which were aneuploid, with the remaining 3,224 (47.9%) normal oocytes available for transfer. In all, 41.7% of oocytes had meiosis I errors, compared to 35.1% with meiosis II errors. Abnormalities in meiosis I were represented by extra chromatids in 15.4%, missing chromatids in 48.1%, missing chromosomes in 5.9%, extra chromosomes in 0.5%, and complex abnormalities in 30.1%. The proportions of abnormal oocytes with missing or extra chromatids in meiosis II were 36.6 and 41.2% respectively, with the remaining oocytes having complex abnormalities, involving missing or extra chromatids of different chromosomes (22.1%) following meiosis II. Overall, 41.8% oocytes had meiosis I, 30.7% meiosis II, and 27.6% both meiotic division errors. A total of 45.1% of the abnormal oocytes had complex errors, involving the same chromosome in both meiotic divisions (21.5%), or different chromosomes (78.5%), of which 74.8% were with abnormalities of two, and 25.2% with abnormalities of three chromosomes studied. Of 3,224 detected aneuploidy-free zygotes, 2,587 were transferred in 1,100 treatment cycles (2.35 embryos per transfer), resulting in 241 (21.9%) clinical pregnancies and 176 healthy children born, suggesting a positive clinical outcome following aneuploidy testing of oocytes in a group of IVF patients of average age 38.5 years.     

Second polar body extrusion is highly predictive for oocyte fertilization as soon as 3 hr after intracytoplasmic sperm injection (ICSI)

Objective Our objective was to evaluate the time course and the predictive value of the extrusion of the second polar body after intracytoplasmic injection (ICSI) related to the fertilization rate, embryo cleavage and quality.Setting The setting was the in vitro fertilization program of a university hospital.Patients Twenty-one patients were treated with intracytoplasmic single sperm injection either for fertilization failure in IVF, low fertilization in IVF (<5%), or severe male factors.Design One hundred thirty-five of 205 metaphase 2 oocytes treated with intracytoplasmic single sperm injection were observed 1, 2, and 3 hr after the assisted fertilization procedure. Extrusion of the second polar body was recorded. For each of these oocytes, fertilization was noted 18 hr after ICSI and cleavage and embryo quality were assessed 24 hr later. The 70 remaining oocytes were used to assess a possible negative effect of repeated exposure to light microscopy.Results The extrusion of the second polar body 3 hr after injection was an observation with a sensitivity of 0.87, a specificity of 0.58, and a high positive predictive value (0.90) toward oocyte fertilization. Twenty-nine and four-tenths percent of the oocytes extruded a second polar body within the first hour, 56.6% within the first 2 hr, and 78.3% had a second polar body 3 hr after injection. This time course was related neither to the speed of embryo cleavage nor to the embryo quality. Fertilization, cleavage, and embryo quality were not affected by repeated observation as deduced from comparison with the control group and confirmed by a high pregnancy (62% per oocyte retrieval) and implantation rate (22% per replaced embryo).Conclusion Oocytes can be checked, in all safety, 3 hr after a single sperm injection for the presence of a second polar to predict oocyte fertilization with a high certainty.     

Cytogenetics of uncleaved oocytes and arrested zygotes in IVF programs

Purpose: Cytogenetic studies of arrested oocytes and zygotes were used to understand in vitro fertilization (IVF) failures. Methods: We investigated the cytogenetics (Giemsa banding and FISH) of 710 uncleaved oocytes and 94 arrested zygotes from 208 patients undergoing IVF procedures. Results and Conclusions: Of uncleaved oocytes without a polar body, 39% were judged cytogenetically abnormal (17% unbalanced predivision and 21.5% diploid). Of 575 oocytes with a polar body, 124 (21.5%) showed numerical or structural chromosome aberrations. In arrested zygotes, approximately equal cases were found with separate condensed haploid complements (no syngamy), nuclear asynchrony and pulverized DNA, and apparently cytogenetically normal zygotes arrested at mitosis. These data on chromosome abnormalities were also analyzed with respect to two ovarian stimulation protocols and to maternal age. Both ovarian stimulation protocols showed the same levels of chromosome abnormalities. Overall chromosome abnormalities and premature chromosome condensation were also unchanged with maternal age. These data illustrate the significance of chromosome aberrations in IVF failures.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

Evidence for maternal predisposition to chromosome aneuploidy in multiple oocytes of some in vitro fertilization patients.

OBJECTIVES: To assess the rate of chromosome aneuploidy (e.g., extra or missing chromosomes) in oocytes remaining unfertilized in our in vitro fertilization (IVF) program. To determine whether two parameters of the IVF technique, advanced maternal age and hormonal follicle stimulation, affect this rate. DESIGN: Data on oocyte retrieval, fertilization, and aneuploidy rates are analyzed to test for possible relations with maternal age and two hormonal stimulation regimens. SETTING: Patients of our IVF program from 119 stimulated cycles over 8 months. PATIENTS, PARTICIPANTS: In vitro fertilization patients selected for having oocytes (1 to 18) remaining unfertilized after insemination in vitro. RESULTS: Advanced maternal age decreases both the number of retrieved oocytes and the fertilization rate, but hormonal treatments have no effect. Aneuploidy (rate 27%), involving group G most frequently, appears associated with advanced age. Patients who were previously parous produced significantly reduced numbers of aneuploid oocytes compared with the nonparous group. A significant excess (P = 0.01) of patients had multiple oocytes all alike (all haploid or all aneuploid), showing correlation among multiple oocytes of a patient in chromosome status. CONCLUSIONS: Maternal age affects reproductive performance and is related to specific chromosomal aneuploidy. Women who were previously parous are more likely to produce normal oocytes than nonparous women; oocyte normality therefore may improve the chance for a future successive pregnancy. Nonrandomness in chromosome abnormality of some patients' multiple oocytes is evidence for maternal predisposition to meiotic nondisjunction. Consequently, these patients are at risk for failed IVF cycles.     

Accuracy of Preimplantation Diagnosis of Single-Gene Disorders by Polar Body Analysis of Oocytes

Purpose: A number of pitfalls in single-cell DNA analysis, including undetected DNA contamination, undetected allele drop out, and preferential amplification, may lead to misdiagnosis in preimplantation genetic diagnosis of single-gene disorders. Methods: Preimplantation genetic diagnosis was performed by sequential first and second polar body analysis of oocytes in 26 couples at risk for having children with various single-gene disorders. Mutant genes were amplified simultaneously with linked polymorphic markers, and only embryos resulting from the mutation-free oocytes predicted by polar body analysis with confirmation by polymorphic marker testing were transferred back to patients. Results: Overall 529 oocytes from 48 clinical cycles (26 patients) were tested, resulting in the transfer of 106 embryos in 44 clinical cycles. As many as 46 (9.6%) instances of allele dropout were observed, the majority (96%) of which were detected. Seventeen unaffected pregnancies were established, of which nine resulted in the birth of an unaffected child, and the rest are ongoing. Conclusions: A high accuracy of preimplantation genetic diagnosis of single-gene disorders is achieved by application of sequential analysis of the first and second polar body and multiplex polymerase chain reaction.     

Correlation between oocyte morphology and the embryo aneuploidy rate in IVF cycles

Abstract

Purpose: To evaluate the aneuploidy rates of 13, 18, and 21 and the X and Y chromosomes in embryos from patients with morphologically normal oocytes and different oocyte dysmorphisms.

Methods: This prospective cohort study included 84 patients treated with in vitro fertilization (IVF) at a single academic center. The patients were divided into the following three groups: group 1 – women with cytoplasmic dysmorphisms (n?=?28), group 2 – women with extracytoplasmic dysmorphisms (n?=?28), and group 3 – women with morphologically normal oocytes (n?=?28). One blastomere from each embryo was analyzed for aneuploidies of chromosomes 13, 18, 21, X, and Y.

Results: The highest prevalence of aneuploid embryos was observed in the group 1 (68.4%) followed by the group 2 (38.9%) and the group 3 (31.3%) (р?<?0.0001). The adjusted OR for receiving an aneuploid embryo in the case of cytoplasmic dysmorphism was 3.6 (95% CI?=?1.8; 7.2), in the case of extracytoplasmic dysmorphisms – 1.3 (95% CI?=?0.7; 2.1).

Conclusions: Women with morphological oocyte abnormalities are at risk for developing aneuploid embryos during IVF cycles. We recommend that woman with cytoplasmic oocyte dysmorphisms receive additional genetic counseling to define the indications for the genetic screening of embryos.     

Ovarian Endometriomas Do Not Adversely Affect Pregnancy Success Following Treatment With In Vitro Fertilization

Purpose: Ovarian endometriomas have an uncertain impact on outcome following in vitro fertilization (IVF). Some authors describe a poor response to ovulation induction, and others observe decreased pregnancy success rates. Conversely, IVF outcomes similar to those of patients undergoing IVF for tubal-factor infertility have also been reported. To determine the impact of ovarian endometriomas on pregnancy success in our IVF program, we identified patients with endometriosis and compared outcomes that were stratified by the presence or absence of an endometrioma at the time of follicular aspiration. Methods: One hundred eight patients with a diagnosis of endometriosis treated with IVF were identified, retrospectively. In this group, 24 patients completed 29 cycles in which an ovarian endometrioma was aspirated at the time of oocyte retrieval, and 84 patients without endometriomas completed 147 cycles. The cycles from these two groups were compared for differences in peak estradiol, number of mature follicles, number of oocytes, number of embryos transferred, and clinical pregnancies. Results: There were no significant differences between the two groups with respect to peak estradiol, mature follicles, number of oocytes, number of embryos transferred, or clinical pregnancies. Conclusions: From this retrospective observational analysis it appears that aspiration of an endometrioma at the time of oocyte retrieval has no adverse effect on outcome. This information may prove helpful when faced with the decision to cancel an IVF treatment cycle in patients with this uncommon complication.     

Detection of aneuploidy in human oocytes and corresponding first polar bodies by fluorescent in situ hybridization

Purpose: The purpose of the study was to investigate the reliability of the fluorescent in situ hybridization (FISH) analysis of the first polar body (IPB) for cytogenetic evaluation of human oocytes as a method of choice in preimplantation diagnosis of chromosomal aneuploidies. Design: Human unfertilized oocytes and their extruded IPB were analyzed using the directly labeled fluorescence alpha-satellite DNA probes to chromosomes X and 18. Results: Paired signals for chromosomes X and 18 were observed in the second meiotic prophase (MII) of unfertilized oocytes and their extruded IPB. In the series of 156 unfertilized oocytes in which the number of X chromosome-and chromosome 18-specific signals were analyzed in both MII and IPB, five nondisjunction events have been detected, with corresponding signals in MII and their IPB: missing signals in MII corresponded to extra signals in their IPB and extra signals in MII corresponded to missing signals in IPB. In one oocyte chromosome 18 nondisjunction was detected, with both chromosome 18 signals in MII and no chromosome 18 signal in IPB. In four oocytes chromatid malsegregations for chromosome X or chromosome 18 were detected: in two oocytes, three of four chromosome 18 signals were present in MII, with only one in IPB, and in the other two oocytes, three of four chromosome signals were present in MII, with only one left in IPB. Conclusions: The data suggest the possibility of detecting chromosomal aneuploidy in oocytes through cytogenetic analysis of their corresponding IPB by FISH as a possible approach for preimplantation diagnosis of major chromosomal trisomies.Presented in part at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, Vienna, Austria, April 3–7, 1995.     

Estimation of Second Polar Body Retention Rate After Conventional Insemination and Intracytoplasmic Sperm Injection: In Vitro Observations from More Than 5000 Human Oocytes

Purpose : Tripronucleate (3pn) development after conventional insemination (CONV) or ICSI was analyzed to estimate the rate of second polar body retention giving rise to 3pn formation. Methods : Data from 453 consecutive IVF cycles were reviewed during a 6-month period. Mature oocytes were monitored in ICSI (n = 3195) and CONV (n = 2274) groups by fertilization assessment 16–18 h post-insemination. Ovulation induction protocols and in vitro culture conditions remained constant during the study interval. Results : Normal (2pn) fertilization occurred in 74.2% and 70.5% for CONV and ICSI groups, respectively (p < 0.003). 1pn formation was observed in 4.5% of CONV oocytes, and 2.5% of ICSI oocytes (p < 0.001); 3pn formation was 8.1% in the CONV group, and 2.5% in the ICSI group (p < 0.0001). We observed 4pn formation in 0.4% of oocytes in the CONV group, but in only 0.04% of oocytes fertilized with ICSI (p < 0.007). Cellular degeneration occurred in 2.4% of oocytes inseminated conventionally, and in 3.5% of oocytes fertilized by ICSI (p = 0.02). Maternal age did not impact pronuclear status. Conclusions : We found the 3pn formation rate after ICSI to be approximately one-third that observed in the CONV group. Extrapolating the ICSI data to the CONV data, it may be inferred that 2.5% of 3pn development after CONV was due to second polar body retention. This suggests that 5.6% of CONV oocytes showed dispermic fertilization. Decreasing oocyte quality with increasing maternal age had no apparent influence on any of the fertilization outcomes.     

Polar body fragmentation in IVM oocytes is associated with impaired fertilization and embryo development

Purpose

The significance of finding a fragmented first polar body in an oocyte prepared for ICSI is controversial with most recent publications suggesting that it is not prognostic for oocyte fertilization or embryo development. Our purpose was to look at this question in the context of oocytes not stimulated for conventional IVF.

Methods

Oocytes obtained for IVM and obtained from follicles at most 12 mm in diameter were evaluated for their polar body morphology soon after they entered metaphase II when they were denuded in preparation for ICSI. Records were evaluated retrospectively for the fertilization rate and the embryo growth rate (cell number) on each day of development for embryos with normal appearing polar bodies or fragmented polar bodies, but no other cytoplasmic dysmorphisms.

Results

Oocytes with fragmented polar bodies were significantly less likely to fertilize than oocytes with normal appearing polar bodies (p < 0.0001). Embryos which developed from oocytes with fragmented polar bodies had significantly impaired growth compared to embryos that developed from oocytes with normal appearing polar bodies (p = 0.0328).

Conclusions

Fragmented polar bodies likely reflect cytoplasmic incompetence.     

Cytogenetic analysis of spontaneously activated noninseminated oocytes and parthenogenetically activated failed fertilized human oocytes--implications for the use of primate parthenotes for stem cell production

Purpose: Spontaneous parthenogenetically activated noninseminated oocytes and failed fertilized oocytes after ART activated by puromycin were studied to assess cleavage ability and the cytogenetic constitution of the resulting embryos. Methods: Failed fertilized oocytes were exposed to puromycin, and whenever activation occurred, they were further cultured until arrest of development. FISH was used to assess the ploidy of spontaneous (group A) and induced parthenotes (group B). Results: The mean number of oocytes exposed to puromycin and the percentage and type of activation were identical in IVF and ICSI patients. The more frequent types of activation were one or two pronuclei and one polar body suggesting that retention of the second polar body is a common event after parthenogenetic activation. Conclusions: Retention of the second polar body and chromosome malsegregation were observed after parthenogenetic activation, either spontaneous or induced by puromycin. This means that using parthenogenetic embryos for stem cell research will require great care and attention.     

Chromosome 21 Detection in Human Oocyte Fluorescence In Situ Hybridization: Possible Effect of Maternal Age

Purpose: The purpose of this study was to evaluate, among 100 uncleaved oocytes, the incidence of numerical and structural chromosome 21 and X abnormalities and to analyze the influence of various factors, such as in vitro (IVF) indications, follicle stimulation protocols, and women's age. Methods: We investigated 150 uncleaved oocytes from 128 patients after an IVF attempt. After cytogenetic analysis (Giemsa) 100 oocytes (66%) were selected for fluorescence in situ hybridization (FISH). Fluorescent probes for human chromosomes X and 21 were used simultaneously according to standard procedures for their hybridization and detection. Results and Conclusions: We analyzed by the FISH protocol 100 metaphase II oocytes with 22 to 25 chromosomes. Our results demonstrate a high rate of disomy for chromosome 21 in human oocytes. Among them, eight were disomic (8%) and three were nullosomic (3%) for chromosome 21. Only one disomy of chromosome X was noted. The various indications of IVF and the different folliculogenesis stimulating protocols did not seem to influence the results but suggested a correlation between the maternal age and the aneuploidy rate of chromosome 21.     

Impact of quantitative parameters,such as number of retrieved oocytes,number of transferred embryos and availability of surplus embryos for cryopreservation,on clinical pregnancy rates in ART

Abstract

Objective: To estimate the impact of quantitative parameters such as number of retrieved oocytes, number of transferred embryos and availability of surplus embryos for cryopreservation on clinical pregnancies in assisted reproductive technology (ART).

Design: We used the database of fertility clinic on in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles from year 2000 to 2010. Follicular fluids containing oocyte–cumulus complexes were recovered by a single lumen follicle aspiration needle, and IVF or ICSI procedure was used depending on infertility indications. Fertilization determination was performed on the second day. Zygotes were transferred into fresh medium and cultivated until the embryo transfer procedure. According to morphological criteria, the best quality embryos were transferred into uterus on the second or third day. Surplus embryos were frozen by slow freezing or by vitrification. We evaluated only clinical pregnancies that were diagnosed by ultrasonography.

Patients: We allocated all 1161 women cohort into three age groups: 671 women were <35; 397 were 35–40 years old and 93 women were >40. Indications for IVF treatment were as follows: mechanical factor (436 cases), male infertility (361 cases), idiopathic infertility (129 cases), endometriosis (78 cases), immunological infertility (14 cases), anovulation (28 cases), and other indications (28 cases). There were no data about the cause of infertility of 87 patients. The stimulation protocol was composed from gonadotropin hormone stimulation with antagonist or agonist supplementation. The triggering of luteinizing hormone (LH) surge was performed by recombinant human chorionic gonadotropin (hCG). The clinical pregnancy rates were: 42.2% (283/671) for women <35, 31.2% (124/397) for women 35–40-year-old and 16.1% (15/93) for women >40.

Interventions: All materials and methods that we used were based on results from our daily practice in IVF clinic and have no experimental design.

Main outcome measures: We evaluated clinical pregnancies in terms of the number of obtained oocytes, the amount of transferred embryos and the availability of surplus embryos for freezing. The number of retrieved oocytes was divided as follows: 1–5 (group I); 6–10 (group II); ≥11 (group III). The number of transferred embryos was from 1 to 3. Cases at which cryopreservation of embryos was not performed were evaluated as well.

Results: For women <35 and 35–40 years old, the highest percentage of pregnancies was achieved when 11 and more oocytes were obtained. Differences in women under 35 between groups were statistically significant. Statistically significant differences in clinical pregnancies depend on the number of transferred embryos in the age groups of <35 and 35–40. Women <35 and 35–40 years old, with the availability of surplus embryos for freezing, had better chances for pregnancy compared with women, who had no excess embryos for freezing.

Conclusions: The number of obtained oocytes, the number of transferred embryos and the availability of surplus embryos for freezing may serve as predictors of pregnancy rates in ART.