Diagnostic and prognostic value of calcium oscillatory pattern analysis for patients with ICSI fertilization failure

Does calcium oscillatory pattern analysis following heterologous intra-cytoplasmic sperm injection (ICSI) of human sperm into mouse oocytes lead to diagnostic and prognostic information for patients suffering from ICSI fertilization failure?

We found that calcium oscillatory pattern analysis following heterologous ICSI has the strength to reveal, for the individual patient, the most probable underlying reason for low or failed fertilization after conventional ICSI.

Fertilization failure occurs in 1-3% of the couples undergoing conventional ICSI, for whom the mouse oocyte activation test (MOAT) or a similar heterologous ICSI model is the only diagnostic test available to evaluate the oocyte-activating capacity of human sperm cells. The MOAT classifies the patients into three groups: a low (group 1), an intermediate (group 2) and a high (group 3) activating group. In MOAT group 1 patients, a sperm-related deficiency is likely to be the cause of previous fertilization failures, while in MOAT group 3 patients a sperm-related deficiency can most probably be refuted. For MOAT group 2 patients, the result is called inconclusive; hence, both sperm and oocyte deficiencies may still contribute to the previous ICSI fertilization failure.

The calcium-releasing ability of sperm from 26 MOAT patients with a history of zero or low fertilization following conventional ICSI was compared with the calcium-releasing ability of sperm from 4 control patients, with proven oocyte activation potential. Per case an average of 19 mouse oocytes were injected. Calcium imaging started within 5-10 min after ICSI and continued for 2 h.

Human sperm were demembranated with 0.02% lysolecithin for 1 min immediately before heterologous piezo-driven ICSI. For calcium imaging, metaphase II oocytes from B6D2/F1 mice were loaded with fura-2 acetoxymethyl ester. The calcium oscillatory patterns following heterologous ICSI were scored per oocyte and per patient individually based on the presence of calcium spikes and their frequency and amplitude.

For patients with low or high MOAT activating capacity (MOAT group 1 or 3, respectively), calcium analysis confirmed the MOAT result. For patients with a former inconclusive intermediate MOAT activating capacity result (MOAT group 2), no or strongly dissimilar calcium oscillatory patterns were seen, with significantly lower amplitude and frequency compared with control sperm. When the product of the amplitude and the frequency of the calcium traces was compared between the groups, MOAT group 1 and 2 cases differed significantly from MOAT group 3 cases and the control sperm (P < 0.01).

The results of the calcium analysis in mouse oocytes should not be directly extrapolated to human oocytes, since it is well known that human spermatozoa exhibit a greater activating potency in mouse oocytes compared with mouse spermatozoa. Furthermore, not much is known yet about the influence of aberrant calcium oscillatory patterns, such as found in MOAT group 2 patients, on pre- and post-implantation embryo development in the human.

Based on the current calcium oscillatory pattern analysis, we found that the product of calcium spike amplitude with its frequency allowed us to create a new threshold value, which can assist in confirming or refuting, on a single patient base, a sperm-borne activation deficiency. The latter is especially interesting for patients with a former intermediate inconclusive MOAT result (MOAT group 2 patients), for whom calcium oscillatory pattern analysis should be considered.

F.V.M. is holder of an aspirant clinical research mandate by the Flemish foundation of Scientific Research (FWO-Vlaanderen). B.H. is supported by a Ghent University grant (KAN-BOF E/01321/01). P.D.S. is holder of a fundamental clinical research mandate by the same Flemish foundation of Scientific Research (FWO-Vlaanderen).