Are we ignoring potential dangers of in vitro fertilization and related treatments?

By conventional assessments, in vitro fertilization (IVF) is one of the safest medical treatments. But producing new life brings immense responsibilities. Recently, there have been disquieting reports of foetal abnormality after these treatments and here we evaluate the potential risks associated with intracytoplasmic sperm injection (ICSI), embryo freezing and pre-implantation genetic diagnosis. In our opinion, before translating new techniques into practice, more research, particularly in animals,is desirable. In addition, better child follow-up and a fresh approach to regulation are also needed.

Kit ligand and c-Kit are expressed during early human ovarian follicular development and their interaction is required for the survival of follicles in long-term culture.

The receptor tyrosine c-Kit and its cognate ligand, c-Kit ligand (KL, stem cell factor, SCF), are involved in ovarian follicular development in several animal species. We studied the expression of KL and c-Kit using in situ hybridization and immunohistochemistry in donated human ovarian cortical tissue. The KL transcripts were expressed in granulosa cells of primary follicles, whereas the expression of c-Kit was confined to the oocyte and granulosa cells in primary and secondary follicles. We employed an ovarian organ culture using firstly serum-containing and then serum-free medium to study the effects of KL and an anti-c-Kit antibody, ACK2, on the development and survival of ovarian follicles in vitro. Culture of ovarian cortical slices for 7 days resulted in a 37% increase in the number of primary follicles and a 6% increase in secondary follicles. The proportion of viable follicles decreased in all cultures. The addition of KL (1, 10 and 100 ng/ml) into the culture media did not affect the developmental stages of the follicles or the proportion of atretic follicles. Inclusion of ACK2 (800 ng/ml) in the culture medium significantly increased the proportion of atretic follicles on days 7 (49 vs 28% in control cultures) and 14 (62 vs 38%) of culture. In conclusion, c-Kit and KL are expressed in human ovaries during follicular development. Blocking the c-Kit receptor induces follicular atresia. The KL/c-Kit signaling system is likely to control the survival of human ovarian follicles during early follicular development.

Quantitative RT-PCR reveals tuberous sclerosis gene, TSC2, mRNA degradation following cryopreservation in the human preimplantation embryo.

The use of cryopreserved human embryos in gene expression studies provides an additional source to the scarce embryos available for research. To validate their use we have implemented a quantitative RT-PCR to characterize the levels of the tuberous sclerosis, TSC2 gene in fresh and frozen-thawed human embryos. Frozen embryos were thawed using two different clinical protocols. In fresh embryos 9.95 fg of TSC2 cDNA was present in the unfertilized oocyte, which was comparable to the level on day 2 of preimplantation development. On day 3 there was a significant drop (P<0.001) to 6.8 fg, followed by an increase in cDNA levels to 10.8 fg (P<0.01) on day 6 at the expanded blastocyst stage. Day 2 frozen embryos possessed 50% less (P<0.001) TSC2 mRNA in comparison to the fresh embryos using thawing protocol one (from frozen to 37 degrees C) and 25% less TSC2 mRNA (P<0.01) with thawing protocol 2 (from frozen to room temperature). After culturing day 2 frozen embryos for an additional day they showed mRNA levels comparable with fresh day 3 embryos. There was no significant difference in the levels of TSC2 mRNA between fresh and frozen day 3 human embryos with either thawing protocol. This study demonstrates that cryopreservation does affect the normal pattern of gene expression during human preimplantation development, and that intact frozen-thawed embryos are not equivalent to their non-frozen counterparts. Furthermore human embryos frozen on day 2 appear to be more susceptible to temperature change than embryos frozen on day 3.

Maintenance of the inner cell mass in human blastocysts from fragmented embryos.

The degree of fragmentation during early cleavage is universally used as an indicator of embryo quality during human in vitro fertilization treatment. Extensive fragmentation has been associated with reduced blastocyst formation and implantation. We examined the relationship between early fragmentation and subsequent allocation of cells to the trophectoderm and inner cell mass in the human blastocyst. We retrospectively analyzed data from 363 monospermic human embryos that exhibited varying degrees of fragmentation on Day 2. Embryos were cultured from Day 2 to Day 6 in Earle balanced salt solution with 1 mM glucose and human serum albumin. Rates of development and blastocyst formation were measured. The number of cells in the trophectoderm and inner cell mass and the incidence of apoptosis were assessed following differential labeling with polynucleotide-specific fluorochromes. Increasing fragmentation resulted in reduced blastocyst formation and lower blastocyst cell numbers. For minimal and moderate levels of fragmentation, the reduction in cell numbers was confined largely to the trophectoderm and a steady number of inner cell mass cells was maintained. However, with extensive fragmentation of more than 25%, cell numbers in both lineages were reduced in the few embryos that formed blastocysts. Apoptotic nuclei were present in both the trophectoderm and inner cell mass, with the lowest incidence in blastocysts that had developed from embryos with minor (5-10%) fragmentation. Paradoxically, higher levels of apoptosis were seen in embryos of excellent morphology, suggesting a possible role in regulation of cell number.

Resultados de tratamento com fertilizaçåo in vitro e transferência de embriöes em pacientes com endometriose / results of in vitro fertilization for the treatment of patients with endometriosis

Os resultados da literatura sugerem que o resultado do tratamento com FIV-TE para pacientes com endometriose depende do estadiamento da doença, sendo que pacientes com endometriose severa têm uma elevada taxa de insucesso. A presença de abortamento é supostamente mais prevalente em mulheres em tratmento para endometriose. Cento e quarenta pacientes com endometriose submeteram-se a 182 ciclos de FIV-TE utilizando a GnRH. Pacientes com endometriose como única patologia foram alocadas em um grupo, e os resultados foram comparados com outros três grupos de pacientes submetidas ao mesmo tratamento, durante o mesmo período. O gupo 1 era composto de casais com fator masculino (45 ciclos);grupo 2, casais com ESCA (196 ciclos), e grupo 3, casais com fator tubário como única causa (1.139 ciclos). A idade média das pacientes, o número médio de ampolas de HMG utilizads, os níveis séricos de E2 no dia do ECG, o número de dias de HMG, o número médio de oócitos aspirados e a taxa de aspiraçäo näo foram significativamente diferentes. A taxa de fertilizaçäo foi significativamente inferior no grupo 1, sendo que nenhuma diferença foi observada entre osdemais três grupos. O número médio de embriöes normalmente fertilizados näo foi significativamente diferente. O número de embriöes transferidos por ciclo e a taxa de implantaçäo foi semelhante nos quatro grupos. A taxa de gravidez por TE foide 39 por cento no grupo 1;48 por cento no grupo 2;45 por cento no grupo 3 e 40 por cento no Grupo 4. A taxa de abortamento foi de zero para o grupo 1; 3,9 por cento para o grupo 2; 3,9 por cento para o grupo 3 e, diferente do exposto em todos os artigos prévios, nenhuma das pacientes grávidas e com endometriose apresentou abortamento. O grupo de endometriose foi dividido de acordo com a classificaçäo do AFS-r e analisado separadamente. Nenhuma diferença foi encontrada nos resultados entre as pacientes com estádio I-II e III-IV. Nossos resultados sugerem que a utilizaçäo de GnRH para supressäo hipofisária melhora os resultados de FIV-TE em pacientes com endometriose. A presença de endometriose e seu grau de severidade näo alteraram significativamente os resultados de FIV-TE, além disso näo encontramos evidências de elevaçäo nas taxas de abortamento espontâneo