PGD周期中机械法和激光法活检对胚胎发育及妊娠结局的影响

目的:比较机械法和激光法进行卵裂球和极体活检对胚胎发育及着床前遗传学诊断(preimplantation genetic diagnosis,PGD)周期妊娠结局的影响。方法:本研究包括20对夫妇的21个PGD周期,其中18个周期分别用机械法或激光法于受精后第3天进行卵裂球活检,并用荧光原位杂交(fluorescence in situ hybridization,FISH)分析检出的卵裂球,于受精后第5或第6天移植信号正常的胚胎;2个周期分别用机械法和激光法在取卵后行极体活检,后行胞浆内单精子注射(introcytoplasmic sperm injection,ICSI)。同时将活检取出的极体进行FISH分析,于取卵后第3天移植经FISH检查正常的卵发育而来的胚胎。另外一个周期先用激光法实行了极体活检,由于FISH检查均无信号,后又用激光法对胚胎行卵裂球活检。结果:共活检胚胎145枚,其中109枚用机械法,36枚用激光法。活检后胚胎的继续发育率分别为72.48%和83.33%,囊胚形成率为33.94%和44.44%,临床妊娠率为38.46%和16.67%,着床率为21.43%和8.33%,两种方法无显著差异。对27枚卵行极体活检,其中12枚用机械法,15枚用激光法。活检后2PN受精率分别为58.33%和46.66%,继续发育率为66.67%和60.00%,亦无显著差异。对活检出的极体进行FISH分析,用机械法活检的极体信号阳性率为90.00%,显著高于激光法的28.57%。结论:用机械法和激光法行极体或卵裂球活检对胚胎发育的影响差异无统计学意义。但使用机械法活检卵裂球能获得较高的临床妊娠率和着床率。极体活检时能获得较高的受精率和继续发育率,故推荐使用机械法进行活检。     

Laser-assisted human embryo biopsy on the third day of development for preimplantation genetic diagnosis: two successful case reports

OBJECTIVE: To perform preimplantation genetic diagnosis (PGD) with 1.48-microm infrared diode laser assistance during embryo biopsy for two patients undergoing IVF. DESIGN: Case reports. SETTING: Private ART laboratory.Two couples undergoing IVF for infertility therapy, both of whom had previously delivered offspring afflicted with spinal muscular atrophy (type 1) after IVF therapy, and who underwent subsequent cycles of IVF coupled with PGD to screen for this disorder. INTERVENTION(S): Two individual IVF cases involving intracytoplasmic sperm injection (ICSI), embryo biopsy with laser assistance, and PGD.The ease and apparent safety of human embryo biopsy using a 1.48-microm infrared laser for partial zona pellucida (ZP) dissection to assist with embryo blastomere biopsy was evaluated. RESULT(S): Both couples were deemed to have some unafflicted embryos for transfer on the fifth day of development after blastomere biopsy in conjunction with PGD. Patient A had a singleton pregnancy and delivered a healthy normal singleton male. Patient B had a twin pregnancy; however, one twin was spontaneously lost at 10 weeks but she ultimately delivered a healthy normal singleton male. CONCLUSION(S): These successful outcomes help to demonstrate the efficacy and safety of laser-assisted embryo biopsy to facilitate PGD screening.     

Preimplantation Diagnosis of Common Aneuploidies by the First- and Second-Polar Body FISH Analysis

Purpose: A low pregnancy rate in in vitro fertilization (IVF) patients of advanced maternal age may be caused by aneuploidies originating from non disjunction in the first or second meiotic divisions. We introduced genetic testing of oocytes by sampling and fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies, to avoid fertilization and transfer of aneuploid oocytes in IVF patients of advanced maternal age. Methods: Three hundred and sixty-three IVF patients 34 years and older participated in the study. Using micromanipulation procedures, the first and second polar bodies were removed following their extrusion from the oocytes and studied by FISH, using probes specific for chromosomes 13, 18, and 21 to detect oocytes with common aneuploidies. Results: Of a total of 538 IVF cycles, 3250 oocytes were available for FISH analysis, with conclusive FISH results in 2742 oocytes (84.3%). As many as 1102 (40%) of oocytes were predicted to be aneuploid and not transferred. Of 1640 embryos predicted to be normal, 1145 were transferred in 467 treatment cycles, resulting in 107 pregnancies (23%), from which 67 healthy children have been born, 32 pregnancies spontaneously aborted, and 15pregnancies are ongoing after being confirmed normal by prenatal diagnosis. Conclusions: Preimplantation diagnosis by first- and second-polar body FISH analysis allows us to avoid the age-related risk of common aneuploidies in IVF patients of advanced maternal age.     

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     

Vitrification of human single pronuclear oocytes following two approaches to polar body biopsy

The purpose of the present study was to investigate whether the size of the opening in the zona pellucida (ZP) of human single pronuclear (1PN) oocytes made by laser and partial zona dissection (PZD) techniques might interfere with the survival and subsequent development to blastocyst stage upon vitrification and warming. Moreover, the viability of these blastocysts was evaluated by comparing their total cell number (TCN) to the TCN of blastocysts developed from control non-vitrified zona-intact 1PN oocytes. Prior to vitrification, a total of 97 and 88 1PN oocytes were subjected to polar body biopsy using laser-assisted and PZD techniques, respectively. The size of ZP opening made by laser and PZD techniques did not interfere with survival (94.8% and 95.4%) or development to the blastocyst stage (27.8% and 26.1%). However, the TCN of laser-derived blastocysts was significantly lower than the TCN of blastocysts developed from non-vitrified control 1PN oocytes (48.7 ± 3.4 versus 70.8 ± 7.1, P < 0.028). The vitrification protocol used here is thus revealed to be an effective method for cryopreservation of 1PN oocytes following polar body biopsy. However, the viability of blastocysts developed from laser-treated 1PN oocytes seems to be negatively affected by this method of biopsy.     

体外受精-胚胎移植周期透明带透亮致密患者的受精与临床结局

目的：探讨卵子透明带透亮致密患者行常规体外受精（IVF）或卵胞质内单精子显微注射（ICSI）后受精、胚胎发育情况及临床结局。方法：回顾性分析体外受精过程中卵子出现透明带透亮致密的43个周期,其中IvF周期27例,ICSI周期16例,比较受精率与正常受精比例、胚胎质量、临床妊娠结局。结果：27例IVF周期均未受精,行早期补救ICSI,其中23例完全不受精。补救后IVF总体受精率达64．83％,正常受精率59.32％;16例ICSI周期均受精,受精率和正常受精率分别为85．11％和79．01％,均显著高于IVF组（P〈0．05）。IVF组和ICSI组卵裂率分别为97．96％、95．65％,优质胚胎率分别为52．67％、43．75％,组间差异无统计学意义（P〉0．05）。IVF组妊娠率为37．04％,种植率为33．33％;ICSI组妊娠率为31．25％,种植率为25．00％,组间临床结局相近,无统计学差异（P〉0．05）。结论：透明带透亮致密患者的自然受精能力下降,需要采用ICSI方式授精。常规IVF受精失败后采用早期补救ICSI或直接行ICSI能明显改善透明带透亮致密患者的受精结局,但不能改善妊娠结局。     

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.     

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.     

Aspects of biopsy procedures prior to preimplantation genetic diagnosis

Today, preimplantation genetic diagnosis (PGD) is offered in over 40 centres worldwide for an expanded range of genetic defects causing disease. This very early form of prenatal diagnosis involves the detection of affected embryos by fluorescent in situ hybridization (FISH) (sex determination or chromosomal defects) or by polymerase chain reaction (PCR) (monogenic diseases) prior to implantation. Genetic analysis of the embryos involves the removal of some cellular mass from the embryos (one or two blastomeres at cleavage-stage or some extra-embryonic trophectoderm cells at the blastocyst stage) by means of an embryo biopsy procedure. Genetic analysis can also be performed preconceptionally by removal of the first polar body. However, additional information is then often gained by removal of the second polar body and/or a blastomere from the embryo. Removal of polar bodies or cellular material from embryos requires an opening in the zona pellucida, which can be created in a mechanical way (partial zona dissection) or chemical way (acidic Tyrode's solution). However, the more recent introduction of laser technology has facilitated this step enormously. Different biopsy procedures at different preimplantation stages are reviewed here, including their pros and cons and their clinical applications. The following aspects will also be discussed: safety of zona drilling by laser, use of Ca2+/Mg2+-free medium for decompaction, and removal of one or two cells from cleavage-stage embryos.     

Preimplantation genetic diagnosis for beta-thalassaemia: the Sardinian experience

OBJECTIVES: To report the experiences on preimplantation genetic diagnosis (PGD) in couples at risk for beta-thalassaemia in Sardinia. METHODS: 23 couples at risk for beta-thalassaemia were included in the PGD programme with a total of 42 cycles performed. Among these, 11 couples were fertile, while the remaining 12 had associated fertility problems. In vitro Fertilization (IVF), PGD and prenatal genetic molecular confirmation protocols and results are reported. RESULTS: All the patients followed the protocol of ovarian stimulation, oocyte retrieval, intracytoplasmic sperm injection (ICSI), embryo biopsy and genetic analysis. A total of 272 oocytes were fertilized in the regular way, and embryo biopsy was performed on 202 embryos. Out of these 202 embryos, 192 (95%) were successful. The genetic diagnosis was performed on 150 embryos (78.1%). Ninety-eight were identified as unaffected and 75 were transferred in 31 cycles. In the infertile patient group, two biochemical pregnancies (11.1% per transfer), in the fertile patient group, four clinical pregnancies, two twin and two singleton pregnancies (30.8% per transfer), were obtained. The genetic molecular results were confirmed in all pregnancies by first-trimester chorionic villus sampling (CVS). CONCLUSION: Our study shows that PGD for beta-thalassaemia is an available procedure for couples who wish to avoid termination of pregnancy, except in cases where the IVF cycle efficiency is very poor.     

Laser-assisted intracytoplasmic sperm injection in human oocytes

OBJECTIVE: To explore potential applications of a non-contact, 1,480-nm diode laser to intracytoplasmic sperm injection (ICSI) of human oocytes. STUDY DESIGN: Human oocytes obtained from in vitro fertilization (IVF) patients and failed to fertilize 24-48 hours after conventional IVF were used for ICSI along with discarded sperm. A noncontact, 1,480-nm diode laser was employed to immobilize sperm, to open a hole in the zona pellucida (ZP) and to perform ICSI through the hole. After ICSI and its simulation, oocytes were examined for formation of pronuclei, cleavage and normality of the cytoskeleton. RESULTS: The 1,480-nm diode laser permitted fast and easy sperm immobilization and microdrilling of ZP to facilitate microinjection. Of the 78 injected oocytes, 53 (68%) survived the procedure, and 13 (25%) of them formed two pronuclei by 18 hours. Further culture of two fertilized eggs resulted in cleavage up to the eight-cell stage before cease of culture. Four oocytes were fixed after simulation of the procedure without injecting sperm. None of them showed gross abnormalities in cytoskeletal organization. CONCLUSION: A noncontact, 1,480-nm diode laser can be used for the immobilization of sperm and for opening a hole in the ZP to facilitate ICSI, biopsy manipulation toward preimplantation genetic diagnosis and assisted hatching.     

Influence of maternal age on the outcome of PGD for aneuploidy screening in patients with recurrent implantation failure

This study assessed the influence of maternal age on the outcome of aneuploidy screening (AS) cycles for recurrent implantation failure (RIF). One hundred and sixteen couples with a history of RIF underwent 130 cycles of AS. Group A included 78 patients aged < or = 40 years (range 25-40 years) who underwent 86 cycles, while group B included 38 couples aged > or = 41 (range 41-47) who underwent 44 cycles. Fluorescence in-situ hybridization (FISH) analysis of the first and second polar bodies using probes specific for chromosomes 13, 16, 18, 21 and 22 was conducted. Euploid oocytes that cleaved were subsequently tested using the same probes on a single blastomere obtained from day 3 embryos. Chromosomally normal embryos were replaced on day 5 of culture. There was no significant difference between the two groups in the mean numbers of oocytes fertilized normally and oocytes (7.5 +/- 3.2 versus 7.2 +/- 3.6) and embryos tested (4.1 +/- 3 versus 3.4 +/-3). However, the younger age group had a significantly higher proportion of euploid oocytes/embryos, cycles reaching embryo transfer, pregnancy (43 versus 25%), clinical pregnancy (36.1 versus 16.6%) and ongoing delivery (32 versus 12.5%) rates per transfer. Preimplantation genetic diagnosis with AS for recurrent IVF implantation failure using FISH probes is therefore associated with improved outcome in women under 41 years, but has a high cancellation rate and low cycle outcome in older women.     

Evidence-based clinical outcome of oocyte slow cooling

In the last few years, there has been a significant improvement in oocyte cryopreservation techniques. To investigate the clinical significance of oocyte freezing, an assessment of the cumulative pregnancy rate per started cycle derived from the use of fresh and frozen-thawed oocytes was performed. Between 2004 and 2006, 749 cycles were carried out, in which no more than three fresh oocytes were inseminated either by standard IVF or microinjection. Supernumerary mature oocytes were cryopreserved by slow cooling. Cryopreservation of fresh embryos was performed in rare cases to prevent the risk of ovarian hyperstimulation syndrome using a standard embryo freezing protocol. Fresh embryo transfer cycles totalled 680, 257 of which resulted in pregnancy. The pregnancy rates per patient and per transfer were 34.3% and 37.8% respectively. When frozen-thawed oocytes were used, following 660 thawing cycles, 590 embryo transfers were performed in 510 patients. Eighty-eight pregnancies were achieved with embryos from frozen oocytes, with a success rate of 17.2% per cycle. When fresh and frozen-thawed cycles were combined, the number of pregnancies was 355, giving a cumulative pregnancy rate of 47.4%. Oocyte cryopreservation can contribute considerably to the overall clinical success, ensuring a cumulative rate approaching that achievable with embryo storage.     

Successful in vitro fertilization and pregnancy by micromanipulation with epididymal sperm

Purpose To examine the effectiveness of micromanipulation on the treatment of the patients with obstructive azoospermia, subzonal insemination or partial zona dissection was performed using epididymal sperm of the 23 patients, and the in vitro fertilization and embryo transfer (IVF-ET) outcomes were analyzed.Results Fertilized oocytes were obtained in 11 cycles of the 25 treated cycles, and six pregnancies were established by the subsequent ET. Through the analysis of 13 cycles in which both the micromanipulation and usual IVF insemination were performed, the fertilization rate per oocyte with the micromanipulation [25% (28/113)] was significantly higher than that with the usual insemination method [4% (4/102)]. The micromanipulation was most useful for the treatment of the patients with congenital absence of the vas deferens. Moreover, there was no need to aspirate a large volume of the epididymal fluid, but it was essential to prepare more than 10 mature oocytes retrieved.Conclusions These results indicate that micromanipulation with epididymal sperm is an effective treatment for obstructive azoospermia, and careful preparations of sperm and wellcontrolled ovarian hyperstimulation are necessary for successful IVF-ET.     

Fertilization of human oocytes by sperm from infertile males after zona pellucida drilling

Infertile couples who had failed to achieve fertilization of oocytes in previous trials of in vitro fertilization (IVF) were treated by IVF with zona pellucida drilling. Zona drilling entails use of micromanipulation to introduce a gap in the zona pellucida either mechanically or by localized application of a zona solvent from a microneedle. Ten couples were treated, from whom 63 oocytes were recovered for manipulation. Sixteen eggs were denuded of the cumulus oophorus only, and the remaining 47 eggs were subjected to zona drilling. Of the 16 eggs denuded but not drilled, 4 (25%) were fertilized. Of the 47 oocytes drilled, 31 survived (67%) and 10 of the surviving eggs (32%) were fertilized. The polyspermy rate for drilled eggs that fertilized was high (5/10, 50%), and polyspermic eggs were often penetrated by more than two spermatozoa. The remaining five eggs fertilized after drilling were diploid fertilizations, and in three cases cleavage was followed by embryo transfer, although pregnancies were not obtained. These data indicate that zona drilling has the potential for establishing pregnancies in instances where treatment by standard IVF would fail. In addition, results indicate that the block to polyspermy in human eggs occurs at the level of the zona pellucida.     

Do quantitative birefringence characteristics of meiotic spindle and zona pellucida have an impact on implantation in single embryo transfer cycles?

Objective

The aim of this study was to determine whether quantitative PolScope characteristics of meiotic spindle and zona pellucida could be used as a non-invasive marker to predict implantation success in elective single embryo transfer cycles.

Methods

Quantitative birefringence parameters; including mean retardance, area, length and polar body deviation angle of meiotic spindle and mean retardance and width of inner zona pellucida belonging to 53 transfer oocytes from elective single embryo transfer cycles were retrospectively analyzed. The relevant PolScope features were compared between 20 conception and 33 non-conception cycles.

Results

Meiotic spindle mean retardance, area, length and inner zona pellucida mean retardance and width did not reveal a statistically significant difference between transfer oocytes from conception and non-conception cycles. Deviation angle of the polar bodies was also comparable between the groups. Spindle and inner zona PolScope characteristics of transfer oocytes were not correlated with the maternal age.

Conclusion

Quantitative PolScope features of meiotic spindle and inner zona pellucida can not be used as a non-invasive marker to predict assisted reproductive technology success in elective single embryo transfer cycles.     

High-dose follicle-stimulating hormone stimulation at the onset of the menstrual cycle does not improve the in vitro fertilization outcome in low-responder patients

In an attempt to improve their outcome with in vitro fertilization (IVF), 34 low-responder patients were stimulated with six ampules of follicle-stimulating hormone (FSH) daily starting on day 1 (n = 17) or day 2 (n = 17) of their menstrual cycles. The stimulated cycles showed a mean peak estradiol of 443 +/- 173 pg/mL, mean days of human chorionic gonadotropin of 7.6 +/- 1.4, 2.67 +/- 1.5 preovulatory oocytes per retrieval, and 2.56 +/- 1.3 oocytes per transfer. Three clinical pregnancies resulted after 25 embryo transfer cycles (12%). With paired analysis, we compared 8 patient cycles with prior six ampules of FSH stimulation starting on day 3; all parameters examined showed no significant differences. In a comparison of 22 patient cycles with prior 4 ampules of FSH stimulation on cycle day 3, no significant differences in any parameters were observed except in the higher number of ampules used in the present study. We conclude that high-dose FSH stimulation at the onset of the menstrual cycle does not improve the IVF outcome in low-responder patients.     

The results of aneuploidy screening in 276 couples undergoing assisted reproductive techniques

Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) using sequential in situ hybridization was applied for aneuploidy testing in 276 couples with 282 ART cycles. Patients with advanced maternal age (AMA, n = 147), recurrent implantation failure (RIF, n = 48), repeated early spontaneous abortion (RSA, n = 32) and abnormal gamete cell morphology (AGCM, n = 55) including macrocephal sperm forms or cytoplasmic granular oocytes were included. Embryo biopsy was performed on day 3 in a calcium-magnesium-free medium by using a noncontact diode laser system. After fixation and enzymatic treatment, fluorescent in situ hybridization (FISH) was carried out on 1147 blastomeres with specific probes for chromosomes 13, 16, 18, 21 and 22 for AMA group, 13, 18, 21, X and Y for AGCM group and 13, 16, 18, 21, 22, X and Y for RIF and RSA groups respectively. The overall chromosomal abnormality rate in analyzed embryos was 40.9%, with no significant difference between AMA, RIF and RSA groups (p > 0.05). However, AGCM group presented a higher rate of chromosomal aneuploidies (57.4%) than the other three groups (p < 0.01). A total of 84% biopsied embryos presented cleavage in 24 h and embryo transfer was realized in 278 cycles. In four cycles, no chromosomally normal embryo was found for embryo transfer. A total of 88 pregnancies (31.6%) were achieved, 19.3% resulted in abortion and 63 healthy births were obtained, with a total of 93 babies born. Aneuploidy testing in couples with poor prognosis undergoing ART cycles is a useful tool to increase the chance of ART success. Furthermore, abnormal gamete cell morphology should be considered one of the major indications for PGD in ART programs as high aneuploidy rates were observed in this group.     

Monozygotic pregnancy after the treatment for infertility by transfer of frozen-thawed embryos]

OBJECTIVES: It has been suggested that monozygotic pregnancies occur more frequently after in vitro fertilisation. This phenomenon is attributed to a number of factors including in vitro culture conditions, malformation of the zona pellucida due to manipulation on the oocytes and artificial opening of the zona pellucida. DESIGN: Retrospective analysis of monozygotic pregnancies in an IVF-ET procedures and obstetrical these pregnancies outcome. MATERIALS AND METHODS: A total of 2254 IVF-ET procedures were analysed. Three protocols were used for ovarian stimulation: short or long protocols with gonadotropins releasing hormone analogue or clomiphene citrate with hMG. In 811 cases male factor was diagnosed and intracitoplasmatic sperm injections were performed. RESULTS: 549 clinical pregnancies were achieved in analysed group. In six cases ultrasound examination 5 weeks after embryo transfer showed a greater number of foetus than the number of embryo transferred. In three of those cases the embryos were obtained after ICSI. Age and average thickness of zona pellucida were similar in group of patients with monozygotic pregnancies when compared with all pregnant patients after IVF treatment. CONCLUSIONS: The incidence of monozygotic pregnancy is increased in group pregnancies resulting from IVF ET. No single risk factor can explain this phenomena. This type of pregnancy needs special obstetrical attention.     

IVF-patients with nonmale factor "to ICSI" or "not to ICSI" that is the question?

PURPOSE: Intracytoplasmic sperm injection (ICSI) guarantees high fertilization rates and could theoretically lead to higher implantation rates as well. Furthermore injection into oocyte creates a hole in the zona pellucida similar to the procedure of assisted hatching. We were therefore interested to assess such a potential benefit for infertile IVF patients without male factor. MATERIALS AND METHODS: Open randomized prospective study according to the rules "Good Clinical Practice" with informed consent of the patients and institutional review board approval. Ninety-one consecutively seen patients with tubal infertility or hostile cervical mucus were randomized to undergo either ICSI (44 patients) or IVF (45 patients). In two patients fertilization of oocytes failed and so a repeated ICSI had to be performed. All these patients were stimulated with the same protocol, using the gonadotropin releasing hormone-agonist (GnRH-a) buserelin acetate in an ultrashort flair-up protocol together with pure follicle stimulating hormone (rFSH). The two study groups did not differ in terms of age, BMI, and all baseline hormone levels. RESULTS: The total pregnancy rate was 42% in the normal IVF group with 33% ongoing pregnancies. The ICSI group had a total pregnancy rate of 39% with 23% ongoing pregnancies. The implantation rate per transferred embryo was higher for normal IVF but not significant (18% versus 11%). The variables, fertilization rate, age, body mass index, baseline hormone levels, endometrial thickness, embryo score, and the highest grade embryo per transfer were very similar in both groups. CONCLUSION: ICSI should be applied only when conventional IVF fails, that is, for male factor patients and for patients with unexplained infertility.