A nivel provincial y local

3.6h (i) Timing of different cleavage stages

The earliest and latest times that each of the cleavage stages was

observed in non-arrested parthenote embryos are show n in table 3.12. The tim ing of each stage of developm ent is broadly sim ilar to that seen in fertilised embryos (plate 3.5).

T a b le 3.12

Tim ing of different stages of development in human parthenogenetic embryos

S ta g e o f d e v e lo p m e n t E a rlie s t s e e n [Hours post activatio n ] L a te s t s e e n [Hours post activatio n ] Extrusion of 2nd polar body 5.5 7.5 P ro n u c le i o b s e rv e d 7 25 C le a v a g e 2 cell 18 48 3 cell 25 44 4 cell 38 64 5 cell 41 69* 6 cell 46* 87* 7 cell 64* 65* 8-10 cell 64* -

Apart from parthenotes that were fixed shortly after an observation (*), figures refer to activated oocytes or parthenote embryos that underwent at least one further stage of developm ent after the recorded observation, and were not therefore arrested.

HOURS POST ACTIVATION

Plate 3.5

Pictorial representation of timing of various developmental events for human parthenogenetic embryos, described in 3.6a and 3.6b(i)

3.6b (ii) Cleavage rates

Of the total 363 activated oocytes, 96 w ere fixed or labelled betw een 5 and 30 hours post activation and w ere therefore excluded from consideration w ith regard to cleavage. 167 of the rem aining 267 activated oocytes allowed to continue in culture exhibited at least one round of cell division, giving an overall cleavage rate of 63% (table 3.13; plates 3.6 - 3.9). The cleavage rates of the different m edia types varied betw een 54% and 80%, b u t these

differences w ere not significant (table 3.13).

The cleavage rates in different types of parthenogenetic activation w ere exam ined, in particular to determ ine w hether diploid 2PN parthenotes w ere m ore likely to cleave than haploid IP N parthenotes. H ow ever com parison of cleavage rates for 1 and 2PN parthenotes (63% and 71% respectively) show ed no significant difference p=0.29) (table 3.13). Im m ediate cleavage oocytes w ere judged to have cleaved if they exhibited further division. The ongoing cleavage rate in this group w as low (28%), and w as significantly lower than that for IP N (%^=10.8, p=0.001) or 2PN (X^=11.6, p=0.0007) parthenotes. Thus it w ould appear that although higher activation rates w ere observed in Medi-Cult, this benefit m ay be offset by a higher proportion of im m ediate cleavage parthenotes w ith poor

developm ental potential.

3.6b (in) Fragmentation

While observing cleavage stage parthenote embryos it w as noticed that cleavage w as often asymmetrical producing irregularly sized blastom eres and fragm entation w as common. Overall 62% parthenote em bryos exhibited some degree of fragm entation (table 3.13).

Table 3.13

Cleavage rates of parthenogenetically activated oocytes allowed to progress in culture beyond 30 hours post activation (hpa).

Medium EBS+HIS EBS+ALB T6+HIS Medi-Cult Total

Total no. activated 124 34 34 171 363

No. fixed or used <30hpa 42 10 14 30 96 Total allowed to continue in culture 82 24 20 141 267 Cleavage rate (%) by type of activation

OPN but cleaved 8/8 (100%) 1/1 (100%) 6/6 (100%) 7/7 (100%) 22/22 (100%)

1PN 32/59 (54%) 12/20 (60%) 7/9 (78%) 46/67 (69%) 97/155 (63%)

2PN 9/10(90% ) 0/3 (0%) 2/2 (100%) 19/27 (70%) 30/42 (71%)

3PN 3/5 (60%) - 0/1 (0%) 5/7 (71%) 8/13(62% )

>4PN - - 1/1 (100%) 1/5 (20%) 2/6 (33%)

immediate cleavage (i.e. further cieavage)

- - 0/1 (0%) 8/28 (29%) 8/29 (28%)

Total cleavage rate 52/82 (63%)

a 13/24 (54%) b 16/20 (80%) c 86/141 (61%) d 167/267 (63%) Fragmentation rate 25/52 (48%) 7/13 (54%) 8/16 (50%) 75/106 (71%) 115/187 (62%)

S ta tis tic s : * = significantly different at th e 5% level

c le a v a g e rate s for different _%2 p value clea v ag e ra te s for different _%2 p value culture m edia groups ty p e s of activation

a & b _0.7 0.41

a & c _2.0 0.16 1 P N & 2 P N _1.1 0.29

a & d _0.1 0.72 1 P N & 3 P N _0.1 0.82

b & c _3.2 0.07 1PN & >4PN _1.0 0.31

b & d _0.4 0.53 1 P N & im m e d iate clea v ag e _10.8 0.001 * c & d _2.7 0.10 2PN & im m ediate clea v ag e _11.6 0.0007

3.6b (iv) Cleavage arrest

As a large proportion of parthenotes (201/363=55%) w ere fixed for analysis of their DNA content, radiolabelled or disaggregated in the first 72 hours after activation, it is difficult to draw definite conclusions about the proportion exhibiting developm ental arrest. Of those parthenote em bryos (n=162) allowed to continue in culture, all arrested by day 5 after

activation, and m ost (93%) by the 2-4 cell stage (table 3.14). A lthough none form ed blastocysts, 9 (6%) parthenotes reached the 6-8 cell stage.

Table 3.14

Developmental stage reached when activated oocyte either a) used for fixation, disaggregation or labelling before definitely arrested or b) allowed to continue in culture until no change in morphology or degeneration observed over > 24 hours (arrested). Developmental stage Non-arrested at fixation/ labelling/ disaggregation Allowed to continue in culture until no change for

>24 hours (arrested) Total Pronucleate or immediate cleavage 96 (48%) 88 (54%) 184 (51%) 1 cell + fragments 5 (2.5%) 7 (4%) 12 (3%) 2 cell 37 (18%) 26 (16%) 63 (17%) 3 cell 22 (11%) 21 (13%) 43 (12%) 4 cell 28 (14%) 10 (6%) 38 (10.5% ) 5 cell 8 (4%) 1 (1%) 9 (2.5%) 6 cell 3(1.5% ) _{1 (1%)} 4(1% ) 7 cell 2(1% ) 4 (2.5%) 6 (2%) 8-10 cell - 4 (2.5%) 4(1% ) Total 201 (100%) 162 (100%) 363 (100%)

Plate 3.6

A two cell human parthenogenetic embryo at 26 hours post activation (hpa)

Plate 3.7

n

Plate 3.8

A four cell parthenote embryo in the process of undergoing cell cleavage (arrow) at 48 hpa.

Plate 3.9

Considering all activated oocytes together 63% com pleted the first cell cycle to 2 cells, 39% attem pted the second round of cleavage to at least 3-4 cells and 9% achieved a third cycle of cleavage to 5-10 cells (table 3.14). 37% (100/267) either failed to cleave at all after the pronucleate stage or only cleaved to a single blastom ere w ith fragments. There did not appear to be any difference in the likelihood of embryonic arrest w hen com paring 1 pronucleate w ith 2 pronucleate parthenogenetic embryos.

3.7 Discussion