Assisted hatching em reprodução assistida: uma meta-análise de ensaios clínicos controlados / Assisted hatching in assisted reproduction: a meta-analysis of clinical controlled trials

O objetivo desta meta-análise foi avaliar o efeito da assisted hatching (AH) sobre os resultados dos ciclos de reprodução assistida: gravidez clínica, nascimento vivo, gestação múltipla, aborto e implantação embrionária, sendo avaliados os artigos publicados em periódicos indexados ao PubMed por dois autores independentes. Foram levantados 51 ensaios clínicos controlados que avaliaram o efeito da AH, sendo 40 excluídos, resultando em 11 artigos completamente avaliados. Não houve diferença significativa na taxa de gestação clínica (44,41 versus 41,30%; p=0,19, AH versus controle, respectivamente) e na taxa de nascimento vivo (36,33 versus 34,79%, p=0,63), porém, foi identificada uma tendência de aumento na taxa de gestação múltipla (18,44 versus 15,02%, p=0,05). Também não foi identificada diferença significativa na taxa de aborto (6,66 versus 6,21%, p=0,83), mas observou-se um aumento significativo na taxa de implantação embrionária (24,32 versus 21,23%, p=0,02). A partir desses resultados, pode-se concluir que, até o momento, não existe evidência suficiente para suportar o uso da AH de rotina para ciclos de reprodução assistida com transferência de embriões frescos, uma vez que não houve aumento na taxa de gravidez clínica e/ou na taxa de nascimento vivo.

The aim of this meta-analysis was to evaluate the effect of assisted hatching on the outcome of assisted reproduction cycles: clinical pregnancy, live birth, multiple pregnancy, abortion and embryonic implantation, by assessing articles published in journals indexed in PubMed by two independent authors. Fifty-one controlled trials that evaluated the effect of assisted hatching were analyzed, and 40 of them were excluded, resulting in 11 articles fully assessed. There was no significant difference in clinical pregnancy rate (44.41 versus 41.30%, p=0.19, assisted hatching versus control, respectively), and in the live birth rate (36.33 versus 34.79%, p=0.63), but we identified a trend toward increased rate of multiple pregnancies (18.44 versus 15.02%, p=0.05). We also did not identify any significant difference in the rate of abortion (6.66 versus 6.21%, p=0.83), but a significant increase in the rate of embryo implantation was observed (24.32 versus 21.23%, p=0.02). From these results, we have concluded that, until now, there is not sufficient evidence to support the use of assisted hatching for routine assisted reproduction cycles with fresh embryo transfer, since there has not been an increase in clinical pregnancy rate and/or the rate of live birth.

Intactness of zona pellucida does not affect the secretion of a trypsin-like protease from mouse blastocyst

Assisted hatching (AH), which is known to improve the hatching potential of mammalian embryos, has been used to increase the pregnancy rate in in vitro fertilization cycles. However, the effect of AH on a trypsin-like protease, which is known to be associated with the hatching process, has not been studied. In this study, we evaluate whether the intactness of zona pellucida affects the secretion of a trypsin-like protease from mouse blastocyst. Four- to 8-cell stage mouse embryos were collected at 66- to 68 hr after hCG injection and divided into 3 groups according to the manipulation of zona pellucida. The groups are no treatment (control), drilling of zona pellucida (ZD) and thinning of zona pellucida (ZT). The activity of a trypsin-like protease, blastocyst development and hatching rate were compared among the three groups at 110 and 135 hr after hCG injection, respectively. The protease activity and blastocyst development were not significantly different among control, ZD and ZT groups at 110 and 135 hr after hCG injection, respectively. However, the hatching rate of ZD and ZT groups was significantly higher than that of control group at each time, respectively (p>0.001). Even in the zona pellucida removed embryos, the protease activity did not differ from the control group. In conclusion, the secretion of a trypsin-like protease from mouse blastocyst does not seem to be affected by the intactness of zona pellucida.

Effect of fertility regulating agents on motility and zona-free hamster egg penetration by spermatozoa of bonnet monkey.

Administration of STS-557 (17 alpha-cyanomethyl-17 beta-hydroxyestra 4,9(10)-dien-3-one; 12 mg/monkey daily) for 4 weeks either alone or in combination with 20 Aet-1 (testosterone-trans-4-n-butyl cyclohexyl carboxylate; code CDB 1781; 40 mg/monkey single administration) had no significant effect on motility and zona free hamster egg penetration by spermatozoa of bonnet monkey, but continuation of the treatment for 12 weeks reduced (in one monkey treated with STS-557) or abolished (one treated with STS-557 and two with STS-557 + 20 Aet-1) the motility as well as zona-free hamster egg penetration (by spermatozoa of all treated monkeys). Motility and the ability to penetrate zona-free hamster egg returned to normalcy after 10 weeks of withdrawal of treatments. Active immunization of monkeys with ovine FSH (4 weeks after booster) had no adverse effect on motility of spermatozoa but none of the zona-free hamster eggs was fertilized. The correlation between motility and the capacity to penetrate the zona-free hamster eggs by monkey spermatozoa varies with the treatment. Such correlation was apparent in monkeys treated with STS-557 but not in monkeys immunized with ovine FSH.

Characteristics of monoclonal antibodies against porcine zona pellucida-3 and their functional relevance.

Seven monoclonal antibodies (MAs) against 55 kDa glycoprotein family of porcine zona pellucida (ZP3) reacting with either ZP3 alpha (MA-7, MA-27, MA-28) or ZP3 beta (MA-1, MA-2, MA-11, MA-30) have been described. MA-1, -2, -27, -28 and -30 do not recognize carbohydrate determinants as shown by their reactivity to the deglycosylated (DG) ZP3 alpha and ZP3 beta. Indirect immunoperoxidase studies showed that all MAs reacted with zona pellucida from porcine and monkey ovaries. Only MA-1 and -27 reacted with ZP from rabbit ovary as well, while none of the MAs recognised mouse ZP, MA-7, -11, -27, -28 and -30 inhibited in vitro, the zona lysis by trypsin as well as the binding of ZP3 to sperm membrane vesicle as investigated by ELISA.

Immunodetection of acrosin during the acrosome reaction of hamster, guinea-pig and human spermatozoa

Mammalian sperm acrosomes contain a trypsin-like protease called acrosin which causes limited and specific hydrolysis of the extracellular matrix of the mammalian egg, the zona pellucida. Acrosin was localized on hamster, guinea-pig and human sperm using monoclonal and polyclonal antibodies to human acrosin labelled with colloidal gold. This was visualized directly with transmission electron microscopy, and with light and scanning microscopy after silver enhancement of the colloidal gold probe. Four distinct labelling patterns were found during capacitation and the acrosome reaction in hamster and guinea-pig spermatozoa, and three patterns were found in human spermatozoa. In the hamster, acrosin was not detected on the inner acrosomal surface after the completion of the acrosome reaction, thus correlating with the observation that hamster spermatozoa lose the ability to penetrate the zona after the acrosome reaction. With guinea-pig and human spermatozoa, acrosin was still detected after the completion of the acrosome reaction, thus correlating with the observation that acrosome reacted guinea-pig spermatozoa bind to and penetrate the zona pellucida