Morphological assessment of oocytes, zygotes and embryos in a human in vitro fertilisation programme

Átírás

1 Morphological assessment of oocytes, zygotes and embryos in a human in vitro fertilisation programme Ph.D. thesis Péter Fancsovits Semmelweis University School of Ph.D. Studies Division of Clinical Sciences Advisor: Programme Director: Official Reviewer: Dr. János Urbancsek M.D., Ph.D. Dr. Zoltán Papp M.D., D.Sc. Dr. Elen Gócza Ph.D. Dr. Gábor Gimes MD., Ph.D. Evaluation Committee: Dr. Béla Szende M.D., D.Sc. Dr. Zoltán Tóth M.D., D.Sc. Dr. Zsolt Csapó M.D., Ph.D. Budapest 2006.

2 1. Introduction In vitro fertilisation (IVF) treatment has become a widely used procedure during the last 20 years in most countries. As a result of the more effective ovarian stimulation protocols, and of improved fertilisation and embryo culture techniques, patients may produce more good quality embryos. Nevertheless, the implantation rate of individual embryos still remains low. To increase the pregnancy rates and reduce the occurrence of multiple pregnancies, we need to transfer fewer but more viable embryos. Selecting the most viable embryos is a great challenge in clinically assisted reproduction. Recently, most IVF units select the embryos on the basis of their morphological appearance at the time of embryo transfer (ET). However, many other strategies have been developed and have led to a widening the range of morphological features which refer to embryo viability. It has been reported that embryo quality correlates with zygote or even with oocyte morphology. Thus, oocyte and zygote morphological characteristics can give us some additional data which can be used in selecting the most viable embryos for transfer. On the basis of previous studies regarding oocyte morphology, we can conclude that an ideal oocyte should have an intact first polar body, a clear, moderately granular cytoplasm, a small perivitelline space and a colourless zona pellucida. However, more than half of the oocytes show at least one morphological anomaly. Oocyte anomalies can be divided into extracytoplasmic and cytoplasmic abnormalities. Extracytoplasmic abnormalities include abnormal morphology of the first polar body, enlargement of the perivitelline space, presence of debris within this space, and irregular thickness, shape and colour of the zona pellucida. Cytoplasmic abnormalities include dark cytoplasm, dark incorporations, spots, granules, refractile bodies and vacuoles. On the basis of previous results, it seems that some of the oocyte dismorphisms influence the fertilisation and further embryonic development. However, previous data are conflicting on this topic. The dynamics of development at the early embryonic stage also indicate embryo viability. Several studies pointed out that embryos which had undergone the first cleavage by hours post-insemination had a better viability than those zygotes that had the first cleavage later. It has also been shown that, 3-4 hours before the first cleavage, the membrane of pronuclei broken down and the two pronuclei disappears (pronuclear breakdown, PNBD). It poses the question, whether the observation of PNBD can be used as an additional selection factor in the embryo selection procedure. 2

3 2. Aims After a thorough study of the literature we introduced a detailed oocyte and zygote morphological assessment in our IVF program in October This morphological assessment system allowed us to analyse the different oocyte dismorphisms individually and objectively. Our data were analysed retrospectively with the aims of: 1. Analysing the effect of the abnormalities of the first polar body on fertilisation, cleavage and embryo morphology. 2. Analysing the effect of the size of the perivitelline space in the oocyte on fertilisation, cleavage and embryo morphology. 3. Analysing the effect of different numbers, size and type of cytoplasmic granules on fertilisation, cleavage and embryo morphology. 4. Analysing the effect of different number, size and type of cytoplasmic vacuoles on fertilization, cleavage and embryo morphology. 5. Determining the optimal time of the assessment of pronuclear breakdown in in vitro fertilised human oocytes. 6. Examining the correlation between early pronuclear breakdown and further embryo development and defining whether early pronuclear breakdown can be an additional factor in the selection of embryos for transfer. 3

4 3. Material and methods 3.1. IVF-ET treatments In this retrospective study we analysed the data of IVF treatment performed in the Embryology Laboratory of the Division of Assisted Reproduction, First Department of Obstetrics and Gynaecology, Semmelweis University School of Medicine between October 2001 and February During the study period we performed 795 oocyte collections on 507 infertile patients with the aim of IVF-ET treatment. A total of 6090 oocytes were collected during the treatments. Oocytes were fertilised by conventional in vitro fertilisation or intracytoplasmic sperm injection (ICSI) according to semen quality, indication of treatment, fertilisation result of previous treatments and number of oocytes obtained. In case of ICSI treatment we performed a detailed morphological assessment of oocytes as it is described in Article hours after conventional IVF or ICSI, normal fertilisation was confirmed by the presence of two pronuclei. Early embryonic development was examined hours after insemination or microinjection as it is described in Article 3.3. Two and three days after fertilisation embryos were assessed for cell number, uniformity of blastomere size, and amount of fragmentation. Embryos were transferred on day 2 or 3. Embryo selection was performed on the basis of their morphology on the day of ET. Data of oocyte morphology or the time of pronuclear breakdown was not taken into consideration in the selection procedure. Pregnancy was defined as increasing serum hcg values measured 11 and 13 days after embryo transfer and clinical pregnancy was considered if a foetal sac was visualised on ultrasound. In case of clinical pregnancy the number of implanted embryos was also recorded Morphological assessment of oocytes Oocyte morphology was assessed after removing cumulus cells in ICSI cycles only. Assessments included the morphology of the first polar body, the size of perivitelline space and the occurrence of granules and vacuoles in cytoplasm. Oocytes were classified into five groups according to the morphology of the first polar body as follows: [I] intact, round or ovoid with smooth surface; [II] intact, round or ovoid with rough surface; [III] fragmented or degenerated; [IV] larger than normal (>20 µm); [V] immature, cap-like structure which was not completely separated from the oocyte. Oocytes were classified into groups according to the size of perivitelline space as follows: [I] tight PVS (<5 µm); [II] enlarged PVS (5-10 µm); and [III] considerably enlarged PVS (>10 µm). 4

5 According to the cytoplasmic granularity, oocytes were classified into five groups as follows: [I] oocyte without granularity or with 1-2 small granules (<5 µm); [II] more than 3 small granules; [III] large granules (>5 µm) which have the some colour as the cytoplasm; [IV] refractile body (>5 µm) which is highly refractile and has a darker colour than the cytoplasm; [V] crater-like dense centrally located granularity. Oocytes were classified according to the occurrence of cytoplasmic vacuoles as follows: [I] no vacuoles; [II] 1-2 small (<10 µm) vacuoles; [III] large vacuole (>10 µm) or more small vacuoles; [IV] sacculus-like structure (accumulation of smooth endoplasmatic reticulum [=ser] clusters) Assessment of early embryonic development Normally fertilised oocytes were examined hours after insemination or microinjection to determine if pronuclear breakdown or first cleavage had occurred. Zygotes were categorised into 3 groups according to their developmental stage: [I] 1 cell zygote with two visible pronuclei; [II] 1 cell zygote with disappeared pronuclei; [III] cleaved embryo (two or more cells). In considering the disappearance of pronuclei, zygotes and embryos belonging to groups 2 and 3 were designated as zygotes which have passed pronuclear breakdown early. Those zygotes in which the pronuclei were clearly visible at the time of assessment were considered as late pronuclear breakdown zygotes. Only cycles that led to embryo transfer with embryos of identical stage at zygote evaluation (i.e. pronuclei disappeared or two clearly visible pronuclei at hours post-insemination) were included in the analysis of cycle characteristics, pregnancy and implantation rates Statistical analysis Statistical analysis was performed using the Statistica software package (StatSoft Inc., Tulsa, OK, USA). Student's t-test was used to compare continuous variables and χ 2 -test was performed for the comparison of proportions across groups. Statistical significance was set at P< Results 4.1. Assessment of first polar body morphology A normal sized and shaped, smooth or rough surfaced first polar body was observed in almost 50% of the oocytes, while the other 50% of the oocytes contained a fragmented, degenerated or irregular sized or shaped polar body (Table 1). 5

6 The highest rate of normal fertilisation was observed in oocytes with fragmented PB (group III.), which was significantly higher than in group II. At the same time the fertilisation rate was significantly lower in group IV (oocytes with large PB) than in group I, II and III. The morphological features of the first polar body had no effect on the cell number of cleaving embryos and the frequency of good quality embryos. However, the amount of anuclear fragments was significantly lower in group III (fragmented or degenerated polar body) than in groups I, II and IV (Table 1). Table 1. Effect of first polar body morphology on fertilisation and further embryo development (for classification of oocytes according to first polar body morphology see article 3.2.) Type of first polar body Σ I II III IV V P-value Number of oocytes 3387 (100) Fertilisation rate 2254 (66.5) Cleavage rate 2194 (64.8) Number of blastomeres a 4.1 ±1.3 Amount of fragmentation a 17.1 % ±13.1 Number of good quality 744 embryos (33.9) NS = Not significant a Values are averages (±SD) b Pearson χ 2 test c Kruskal-Wallis test 714 (21.1) 468 (65.5) 459 (64.3) 4.0 ± ± (34.0) 1006 (29.7) 650 (64.6) 628 (62.4) 4.0 ± ± (34.2) 1576 (46.5) 1088 (69.0) 1061 (67.3) 4.1 ± ± (34.4) 62 (1.8) 31 (50.0) 30 (48.4) 4.3 ± ± (16.7) 29 (0.9) 17 (58.6) 16 (55.2) 4.4 ± ± (18.8) b b NS c c NS b 6

7 4.2. Assessment of the size of the perivitelline space Almost 60% of oocytes contained a tight or slightly enlarged perivitelline space, while 40% of oocytes had a considerably larger PVS (Table 2). Fertilisation rate and cleavage rate was not different between groups according to the size of perivitelline space. The fastest development was observed in the embryos originated from oocytes with tight PVS (group I) while enlarged PVS (group III) resulted in embryos with considerably lower cell numbers. The difference between groups was significant. The amount of fragmentation was significantly decreased while the size of perivitelline space was increased. The frequency of good quality embryos was the highest in group III, and it differed significantly from group I. However, the number of good quality embryos in group II was not different from the other groups (Table 2). Table 2. Effect of size of perivitelline space on fertilisation and further embryo development (for classification of oocytes according to the size of perivitelline space see article 3.2.) Size of perivitelline space Σ I II III P-value Number of oocytes (100) (26.5) (32.1) (41.4) Fertilisation rate NS b (67.8) (67.9) (70.0) (66.1) Cleavage rate NS b (65.8) (65.9) (68.4) (63.8) Number of blastomeres a <0.001 c ±1.4 ±1.5 ±1.3 ±1.3 Amount of fragmentation a % <0.001 c ±13.6 ±13.9 ±14.2 ±12.5 Number of good quality b embryos (32.3) (26.7) (31.6) (36.5) NS = Not significant a Values are averages (±SD) b Pearson χ 2 test c Kruskal-Wallis test 7

8 4.3. Assessment of cytoplasmic granulation Five per cent of oocytes had no granularity at all or contained only minimal granularity. Another 45% of the oocytes contained several small granules, while almost 50% of the oocytes had large granules, refractile bodies or dense centrally located granularity (Table 3). Normal fertilisation rate did not differ between groups. Cleavage rate was highest in group I, which was significantly higher than in the group of oocytes with a refractile body. Oocytes with large granules also had a significantly higher cleavage rate than was observed in group II or group IV. Embryo morphology was not different between groups on day 2 (Table 3). However, the amount of fragmentation was significantly higher in the refractile body group (25.1±17.7%) than in group II (19.0±15.1%; P<0.001) and in group III (20.2±16.4%; P=0.006) 3 days after the fertilisation (data not shown). Oocytes with dense centrally located granularity (group V) also resulted in a significantly higher amount of fragmentation (21.9±14.9%) than was observed in group II (19.0±15.1; P<0.002) and group III (20.2±16.4; P=0.026). Table 3. Effect of cytoplasmic granularity of oocytes on fertilisation and further embryo development (for classification of oocytes according to first polar body morphology see article 3.2.) Type of granularity Σ I II III IV V P-value Number of oocytes (100) (5.1) (45.3) (23.9) (10.7) (15.0) Fertilisation rate NS b (67.8) (73.6) (66.2) (71.7) (63.9) (67.4) Cleavage rate b (65.8) (72.8) (64.6) (69.5) (60.8) (64.9) Number of blastomeres a NS c ±1.4 ±1.2 ±1.3 ±1.5 ±1.3 ±1.4 Amount of fragmentation a NS c % ±13.6 ±11.4 ±13.4 ±13.8 ±14.2 ±14.0 Number of good quality embryos 520 (32.3) 36 (39.6) 226 (31.6) 137 (33.7) 44 (27.5) 77 (32.2) NS b NS = Not significant a Values are averages (±SD) b Pearson χ 2 test c Kruskal-Wallis test 8

9 4.4. Assessment of cytoplasmic vacuoles In 90% of oocytes no vacuoles were observed. Almost 5% of oocytes had one or two small vacuoles, while the occurrence of large vacuoles and ser accumulation did not exceed 5% (Table 4). Fertilisation rates and cleavage rates in the groups of oocytes with considerable vacuolisation (group III) or with ser accumulation (group IV) were significantly lower than in the groups with no vacuoles (group I) or minimal vacuolisation (group II). Cell numbers did not differ between groups. Anuclear fragmentation was 17-18% in embryos belonging to groups I, II and III, while in group IV (ser accumulation) significantly lower fragmentation was observed (12.1%). Good quality embryos were observed in a significantly higher number in group IV than in all other groups. Table 4. Effect of cytoplasmic vacuoles of oocytes on fertilisation and further embryo development (for classification of oocytes according to first polar body morphology see article 3.2.) Type of vacuoles Σ I II III IV P-value Number of oocytes (100) (90.0) (5.4) (2.2) (2.5) Fertilisation rate Cleavage rate 2254 (66.5) 2194 (64.8) Number of blastomeres a 4.1 ±1.3 Amount of fragmentation a % 17.1 ±13.1 Number of good quality embryos NS = Not significant a Values are averages (±SD) b Pearson χ 2 test c Kruskal-Wallis test 744 (33.9) 2057 (67.5) 2002 (65.7) 4.1 ± ± (33.5) 121 (66.5) 119 (65.4) 4.2 ± ± (37.0) 33 (45.2) 30 (41.1) 4.0 ± ± (20.0) 43 (51.2) 43 (51.2) 3.8 ± ± (55.8) <0.001 b <0.001 b NS c c b 9

10 4.5. Assessment of early embryonic development Early embryonic development was assessed between hours post-insemination according to the laboratory schedule as described in Article 3.3. Pronuclear breakdown was first observed 21.5 hours after fertilisation. Its frequency did not exceed 20% in the next hour. However, after 25 hours post-insemination pronuclei had disappeared in more than 70% of the oocytes. On the basis of these observations we considered the optimal time of PNBD assessment to be between hours post-insemination. Embryos developed from early pronuclear breakdown zygotes had a significantly higher cell number on day 2 than embryos developed from late pronuclear breakdown zygotes (4.4±1.2 vs. 3.6±1.4; P<0.0001). However, the amount of anuclear fragmentation, and the frequency of good quality embryos was similar in the early and the late PNBD group. The mean cell number of transferred embryos and the average number of top quality embryos transferred was significantly higher in the early PNBD group than in the late PNBD group. However, the amount of fragmentation did not differed significantly between groups. The transfer of early PNBD embryos resulted in a 20% higher clinical pregnancy rate and a 10% higher implantation rate than those with late pronuclear breakdown (Table 5.). Table 5. Comparison of treatment parameters in IVF/ICSI cycles where only early PNBD embryos or only late PNBD embryos were transferred PNBD P-érték Early Late Number of cycles Number of oocytes a 6.7 ± ±3.6 NS b Number of fertilised oocytes a 4.5 ± ±2.6 NS b No. of blastomeres per transferred embryo a 6.7 ± ±2.0 <0.001 b Amount of fragmentation in 12.9 ± ±11.6 NS b transferred embryos a No. of top quality embryos transferred a 1.4 ± ±0.7 <0.001 b Positive hcg test 32/58 (55.2) 46/139 (33.1) c Clinical pregnancy 28/58 (48.3) 38/139 (27.3) c Implantation 41/155 (26.5) 61/404 (15.1) c NS = Not significant a Values are averages (±SD) b Student's t-test c χ 2 -test 10

11 5. Conclusions Using the detailed oocyte and zygote morphological assessment systems which were introduced in our study, we had an opportunity to estimate the effect of different oocytes dysmorphisms on fertilisation and further embryo development of human embryos. 1. Assessment of first polar body Fragmentation of the first polar body has no deleterious effect on fertilisation rate or further embryo quality compared with oocytes containing intact first polar body. Embryos developing from oocytes with a fragmented polar body just as suitable for embryo transfer as embryos developing from oocytes containing intact, normal sized, round or oval polar bodies. The effect of an immature or large polar body cannot be determined easily because of the rare occurrence of these anomalies. However, on the basis of our results, it seems that these oocyte abnormalities considerably diminish the chance of fertilisation and the viability of embryos. Thus, the transfer of embryos developing from oocytes containing these polar body abnormalities should be avoided. 2. Assessment of the size of perivitelline space The size of the perivitelline space does not influence the fertilisation and cleavage rate of oocytes. However, embryos developing from a normal sized perivitelline space have a faster development but lower embryo morphology than embryos developing from oocytes with an enlarged perivitelline space. These contradictory data, compared with previous and also conflicting reports, do not give an acceptable explanation of the effect of the perivitelline space on further embryo development. Further and larger studies are needed in this field to clarify the effect of different sizes of perivitelline space on further embryo development. Observation of implantation rate according to the size of perivitelline space would also be reasonable. 3. Assessment of cytoplasmic granules The occurrence of small or large cytoplasmic granules has no discernable effect on fertilisation and further embryo development. However, refractile bodies and dense centrally located granularity have a detrimental effect on embryo development. This deleterious effect evolves later during preimplantation embryonic development. Thus, we believe that embryos developed from oocytes showing these cytoplasmic anomalies should be avoided in embryo selection process. 11

12 4. Assessment of cytoplasmic vacuoles Occurrence of small vacuoles in oocytes has no effect on fertilisation or further embryo development. However, the presence of considerable vacuolisation or of sacculus-like accumulation of smooth endoplasmatic reticulum (ser) dramatically reduces the chance of normal fertilisation. Developmental data of embryos derived from oocytes with excessive vacuolisation suggests that transfer of these embryos should be avoided. If the presence of ser accumulation does not hinder fertilisation and first cleavage it will not influence the further embryo development. These embryos have better quality than those which developed from oocytes without vacuoles. To make a decision to transfer or not to transfer embryos developed from oocytes with ser accumulation, we suggest further detailed cell biological analysis of these oocytes and embryos. 5. Determining the optimal time of the assessment of early embryonic development On the basis of our results, the optimal time for assessing early pronuclear breakdown is between hours post-insemination. Within this period, the earlier interval is suitable for assessing early pronuclear breakdown in cycles where the number of normally fertilised zygotes is much higher than the number of embryos which we plan to transfer. In case of a lower number of fertilised oocytes the assessment should be performed hours after insemination. Further studies are required to determine the optimal time of pronuclear breakdown assessment in case of IVF and ICSI embryos. 6. Effect of early pronuclear breakdown on the further embryonic development Early pronuclear breakdown results in a faster rate of cell division but its effect on embryo quality cannot be demonstrated on the basis of our results. Transferring embryos with early pronuclear breakdown resulted in a higher pregnancy and implantation rate than transferring embryos which had intact pronuclei at the time of assessment at early embryonic development. This finding suggests that early pronuclear breakdown is as good indicator of embryo viability as early cleavage assessed three hours later. Thus, we suggest the assessment of early pronuclear breakdown, as an alternative to assessment of early cleavage, should be included in the scoring system to select the most viable embryos for transfer. 12

13 6. List of publications 6.1. Journal articles pertaining to the thesis 1. Fancsovits P, Dinnyés A, Dohy J. Emlős embriók in vitro kultivációja (Szemlecikk). Magyar Állatorvosok Lapja 1998;120: IF: 0, Fancsovits P, Bárándi Zs, Tóthné GZs, Urbancsek J. In vitro fertilisatiós kezelések során beültetett praeembryók minőségének hatása a többes terhességek gyakoriságára. Magyar Nőorvosok Lapja 2001;64: Fancsovits P, Bán Z, Tóthné GZs, Urbancsek J, Papp Z. Első lépéseink a praeimplantatiós genetikai diagnosztikában: Blastomera biopsia. Orvosi Hetilap 2001;142: Fancsovits P, Urbancsek J, Papp Z. In vitro fertilisatióval létrehozott praeembryók beágyazódási esélye a petesejt, a zigóta és a praeembryo morfológiai jellemzői alapján. Magyar Nőorvosok Lapja 2002;65: (A közlemény elnyerte a Magyar Nőorvos Társaság és a Magyar Nőorvosok Lapja évi nívódíját) 5. Papp Z, Fancsovits P, Bán Z, Tóthné GZs, Urbancsek J. Előébrény-diagnosztikát követően fogant sikeres terhesség első hazai esete. Orvosi Hetilap 2002;143: Fancsovits P, Toth L, Murber A, Szendei Gy, Papp Z, Urbancsek J. Catheter type does not affect the outcome of intrauterine insemination treatment: a prospective randomised study. Fertility and Sterility 2005;83: IF: 3, Fancsovits P, Toth L, Takacs, FZ, Murber A, Papp Z, Urbancsek, J. Early pronuclear breakdown is a good indicator of embryo quality and viability. Fertility and Sterility 2005;84: IF: 3, Urbancsek J, Fancsovits P, Murber Á, Tóthné G Zs, Hauzman E, Papp Z. In vitro fertilizációs kezelések klinikánkon: Tíz év munkája a számok és eredmények tükrében ( ). Orvosi Hetilap 2006;147: Fancsovits P, Takács FZ, Tóthné GZs, Urbancsek J. A korai praeembryo fejlődés vizsgálata és ennek jelentősége in vitro fertilizációs kezelések során. Magyar Nőorvosok Lapja 2006;69: Fancsovits P, Tóthné GZs, Murber Á, Takács FZ, Papp Z, Urbancsek J. Correlation between first polar body morphology and further embryo development. Acta Biologica Hungarica 2006; (in press) IF: 0,425 13

14 11. Fancsovits P, Takács FZ, Tóthné GZs, Murber Á, Urbancsek J. In vitro fertilisatiós kezelések során nyert petesejtekben megfigyelhető vacuolumok hatása a praeembryók életképességére. Magyar Nőorvosok Lapja 2006; (in press) 6.2. Abstracts pertaining to the thesis, published in international journals 12. Fancsovits P, Bárándi Zs, Fedorcsák P, Tóthné GZs, Papp Z, Urbancsek J. Effect of quality and cell number of transferred embryos on multiple pregnancy rate. Human Reproduction 2000;15S: IF: 2, Fancsovits P, Tóthné GZs, Papp Z, Urbancsek J. Effect of oxygen concentration on embryonic development in vitro. Human Reproduction 2001;16(S1):165. IF: 2, Fancsovits P, Bán Z, Tóthné GZs, Urbancsek J, Papp Z. Blastomere biopsy as the first step in preimplantation diagnosis. Fetal Diagnosis and Therapy 2002;17(S1):31. IF: 1, Fancsovits P, Tothne GZs, Takacs FZ, Papp Z, Urbancsek J. Is there a relationship between the size of perivitelline space of oocytes and fertilization rate, cleavage rate and embryo morphology? Reproductive Biomedicine Online, 2003;7(S1): Fancsovits P, Tothne GZs, Takacs FZ, Papp Z, Urbancsek J. Relationship between cytoplasmic granularity of oocytes and development of human embryos. Human Reproduction 2004, 19(S1):i23. IF: 3, Fancsovits P, Tothne G.Zs, Takacs F.Z, Murber A, Papp Z, Urbancsek J. Effect of enlarged perivitelline space on embryo development. Human Reproduction 2005;20(S1):i90. IF: 3, Fancsovits P, Tóthné GZs, Takács FZ, Murber Á, Papp Z, Urbancsek J. Factors affecting multinucleation in human embryos. Human Reproduction 2006; (in press) IF: 3, Other abstracts pertaining to the thesis 19. Fancsovits P, Bárándi Zs, Tóthné GZs, Hauzman E, Papp Z, Urbancsek J. In vitro fertilisatios kezelések során beültetett praeembryók minőségének hatása a többes terhességek gyakoriságára. Program és absztraktok a MART III. Kongresszusáról. 2001;P Fancsovits P. A petefészekszövet és az ivarsejtek cryopreservatiója és in vitro érlelése. Az ivarsejt és praeembryo bírálati rendszerek szerepe a kezelés kimenetelének előrejelzésében. Nőgyógyászati és Szülészeti Továbbképző Szemle, 2002;4 (S1):

15 21. Fancsovits P, Bán Z, Tóthné GZs, Urbancsek J, Papp Z. Blastomera-biopsia, mint a praeimplantatiós genetikai diagnosztika első lépése. Nőgyógyászati és Szülészeti Továbbképző Szemle, 2002;4(S1): Fancsovits P, Tothne GZs, Papp Z, Urbancsek J. Is there a relationship between cytoplasmic granularity of oocytes and fertilization rate, cleavage rate and embryo morphology? Program and symposium syllabus of ESHRE Campus Symposium, Mammalian oogenesis and folliculogenesis: in vivo and in vitro approaches 2003; P4 23. Fancsovits P, Tóthné GZs, Takács FZ, Papp Z, Urbancsek J. A petesejt citoplazmájában megfigyelhető szemcsézettség hatása a megtermékenyülési és osztódási arányra valamint a praeembryo minőségére. Programok és összefoglalók a MART IV. Kongresszusáról. 2003; Fancsovits P, Tothne GZs, Takacs FZ, Papp Z, Urbancsek J. Does the type of culture media used effect the results of a cryopreservation program? A preliminary study. Program and symposium syllabus of ESHRE Campus Symposium, Cryobiology and cryopreservation of human gametes and embryos. 2004; Fancsovits P, Tóth L, Rusz A, Romics I, Papp Z, Urbancsek J. Cryopreservált hereszövetből származó hímivarsejtek morfológiai eltéréseinek hatása az in vitro fertilisatiós kezelések eredményességére. Program és absztraktok a Magyar Urológusok Társasága, Andrológiai Szekció 29. Tudományos Üléséről. 2005; Fancsovits P, Tóthné GZs, Takács FZ, Murber Á, Papp Z, Urbancsek J. Éretlen (MI) petesejtek megtermékenyítésének lehetősége ICSI kezelések során. Program és absztraktok a MART V. kongresszusáról 2005; Fancsovits P, Takacs FZ, Tothne GZs, Papp Z, Urbancsek J. Correlation between first polar body morphology and embryo quality. Final program and abstracts of Serono Symposia International: How to improve ART outcome by gamete selection. 2005; P Other publications 28. Kissné DDX, Hargitai Cs, Fancsovits P, Péczely P. Seasonal pattern of reproduction of domestic gander (Anser anser). Bulletin Univ. Agric. Sci. 75 th Aniversary. Ed II. 1995; Urbancsek J, Vass Z, Fancsovits P, Hauzman E, Dévényi N, Tóthné GZs, Pohola E. Kezdeti tapasztalataink a GnRH-antagonista cetrorelix in vitro fertilizációs kezelésben való alkalmazása során. Magyar Nőorvosok Lapja 2000;63: Fancsovits P. Kommentár az Alkalmazható-e az emberi klónozás a meddőség kezelésére című cikkhez. Nőgyógyászati és Szülészeti Továbbképző Szemle. 2001;3:

16 31. Bán Z, Fancsovits P, Nagy B, Kamaszné HM, Urbancsek J, Papp Z. Első lépéseink a praeimplantatiós genetikai diagnosztikában: Genetikai vizsgálatok. Orvosi Hetilap 2001;142: Urbancsek J, Hauzman E, Fancsovits P, Murber Á, Pappné RJ, Tóthné GZs, Papp Z. A GnRH-antagonista cetrorelix egyszeri és többszöri adagolási séma szerinti alkalmazásával szerzett tapasztalatok összehasonlítása in vitro fertilisatiós kezelések során. Magyar Nőorvosok Lapja 2002;65: Fancsovits P. Kommentár az Ivarsejtfagyasztás és petefészekszövet-transzplantáció című cikkhez. Nőgyógyászati és Szülészeti Továbbképző Szemle. 2004;6: Hauzman E, Lagarde AR, Nagy K, Fancsovits P, Murber Á, Jánoki Gy, Papp Z, Urbancsek J. Prognostic value of serum CA-125 measurements on stimulation day 1 and on the day of oocyte pickup in the prediction of IVF treatment outcome. Journal of Assisted Reproduction and Genetics 2005;22: IF: 0, Murber Á, Fancsovits P, Urbancsek J. In vitro fertilizációs kezelés késői szövődményeként létrejött adnextorsio a terhesség első trimeszterében. Magyar Nőorvosok Lapja 2005;68: Murber Á, Fancsovits P, Szendei Gy, Urbancsek J. Intrauterin inszeminációs kezelések eredményessége az I. Számú Szülészeti és Nőgyógyászati Klinika Asszisztált Reprodukciós Osztályának 10 éves működése során. Magyar Nőorvosok Lapja 2005;68: Other abstracts 37. Fancsovits P, Bodó Sz, Baranyai B, Dinnyés A, Dohy J. Comparison of defined embryo culture systems in cattle. Theriogenology 1998;49:199. IF: 1, Fancsovits P. LeBourhis D. Heyman Y. Comparison of INRA B2 and Tissue Culture Medium 199 for in vitro production of bovine embryos. Proceedings of the14 th meeting of the European Embryo Transfer Association, 1998; Bán Z. Nagy B. Fancsovits P. Urbancsek J. Papp Z. Sex determination and detection of delta F508 mutation in human blastomeres by F-PCR. European Journal of Human Genetics 2001;9(S1):102. IF: 3, Bán Z, Fancsovits P, Nagy B, Kamaszné HM, Urbancsek J, Papp Z. Sex determination and detection of F-508 mutation in human blastomeres using fluorescent-pcr. Reprod. Toxicol. 2001;15:451. IF: 1,697 16

17 41. Murber Á, Fancsovits P, Tóthné GZs, Hauzman E, Papp Z, Urbancsek J. Effect of catheter type on the outcome of intrauterin insemination cycles. A prospective randomized study. Human Reproduction 2002;17(S1):118. IF: 3, Bán Z, Fancsovits P, Nagy B, Kamaszné HM, Urbancsek J, Papp Z. First attemps in hungary in sex determintation and in detection of delta-f 508 mutation in human blastomeres for preimplantation genetic diagnosis. Fetal Diagnosis and Therapy 2002;17(S1):31. IF: 1, Murber Á, Fancsovits P, Tóthné GZs, Hauzman E, Urbancsek J. Progressive motile sperm count is predictive for treatment outcome in intrauterine insemination cycles. Fetal Diagnosis and Therapy 2002;17(S1):32. IF: 1, Bán Z, Fancsovits P, Nagy B, Kamaszné HM, Urbancsek J, Papp Z. A nemi chromosomák és a cysticus fibrosis delta-f 508 mutáció vizsgálata blastomera-sejtekben. Nőgyógyászati és Szülészeti Továbbképző Szemle, 2002;4(S1): Murber Á, Fancsovits P, Tóthné GZs, Hauzman E, Urbancsek J. A progresszíven mozgó spermiumok száma meghatározó a kezelés eredményessége szempontjából intrauterin inseminatiós kezelések során. Nőgyógyászati és Szülészeti Továbbképző Szemle, 2002;4(S1): Bán Z, Fancsovits P, Gilán Zs, Urbancsek J, Papp Z. First attemps in Hungary at sex determination and detection of F508 mutation in human blastomeres for PGD. Reproductive Biomedicine Online, 2003;7(S1): Murber Á, Fancsovits P, Hauzman E, Papp Z, Urbancsek J. IVF-kezelések során alkalmazott kombinált GnRH-agonista+hMG és GnRH-antagonista+hMG stimulációk összehasonlítása: eset-kontroll tanulmány. Program és absztraktok a MART V. kongresszusáról 2005; Scientific publications: Articles published in international journals: 4 (IF: 7,728) Articles published in Hungarian journals: 15 (IF: 0,073) Abstracts published in international journals: 19 (IF: 29,121) Abstracts published in symposium syllabuses: 9 17