**14. Oocyte maturity**

130 Enhancing Success of Assisted Reproduction

**13. Poor ovarian response** 

discovered until the first IVF cycle.

[135]. Clinical pregnancy and delivery after oocyte activation by electrostimulation in combination with ICSI in previously failed to fertilize oocytes has been obtained [80]. Electrical stimulation rescues human oocytes that failed to fertilize after ICSI and stimulates them to complete the second meiotic division, to form pronuclei and to undergo early embryonic development [136]. Although the fertilization rate issimilar regardless of the number of electrical pulses applied, subsequent embryo development is dramatically improved in those oocytes that received three electrical pulses [136]. The embryo formation rate in the electrically activated group is 80% compared to 16% in the control group [137]. Although the fertilization rate is significantly higher in the electroactivated group (68%) as compared with that of the control (60%), a higher miscarriage rate is reported in the electroactivated group (5 of 15 pregnancies) compared to the control (3 of 33) [6]. Like any other new assisted reproductive procedure, the impact of electrical activation on oocyte and embryo health must be evaluated in larger studies before this procedure can be considered for routine clinical purposes. Ideally, karyotyping or fluorescent *in situ* hybridization analysis should be performed to assess the

The definition of 'poor response' in the literature is often based on a combination of factors, including the number of mature follicles, the number of oocytes retrieved and the peak estradiol level [138]. The cut-off levels for the number of follicles or oocytes that define poor response vary widely from study to study. Some authors feel that the definition of poor response should also include the degree of ovarian stimulation used and that a low oocyte number is detrimental only when high total dose of follicle stimulating hormone (FSH) has been administered [139]. Various endocrine and ultrasonographic markers and dynamic tests to assess ovarian reserve have been evaluated. Such tests include basal FSH on cycle day 3, clomiphene citrate challenge test, inhibin B, oestrogen, anti-Mullerian hormone, antral follicle counts and ovarian volume. The success of each test can be measured against ovarian response or live birth rate per cycle [140]. However, none of these tests has demonstrated a reliable predictive value and for many women poor ovarian response is not

Poor response to gonadotropin stimulation occurs more often in older women, but may also occur in young women, regardless of the endocrinologic profile [141]. Poor responders have a significantly lower pregnancy rate per retrieval compared to normal to high responders in the same age group [138]. Although it is possible to have normal embryos and pregnancy in younger poor responders, the fertilization rate and quality of embryos in older poor responders are always low and the chance of achieving pregnancy in these patients is low. Poor responders also have an increased cycle cancellation rate due to retrieval of few or no oocytes and/or TFF. One of the major contributing factors for TFF after ICSI is ≤3 MII oocytes retrieved (Esfandiari *et al*., 2005a). The rate of fertilization failure increases as the number of injected oocytes decreases [142]. There is a higher chance of having no embryos for transfer and significantly lower pregnancy rates when less than five oocytes are

incidences of aneuploidy and mosaicism in the resultant embryos.

One of the major causes of TFF after ICSI is a low number of retrieved MII oocytes [10]. About 20% of retrieved oocytes from controlled ovarian stimulation cycles are immature, either at metaphase-I (MI) or germinal-vesicle (GV) stage in human IVF [35]. Some of these oocytes may extrude the first polar body during *in vitro* culture and can be injected in ICSI cycles. This may be a useful strategy for patients with low number of retrieved oocytes. However, embryos derived from immature oocytes do not efficiently translate into pregnancies and live births. Therefore, the clinical significance of using immature oocytes in stimulated cycles needs further investigation [148].

The injection of MI oocytes immediately after denudation results in a high degeneration rate due to increased fragility of the oolemma. The fertilization rate of retrieved MI oocytes that remained MI at the time of ICSI is lower than the fertilization rate of sibling retrieved MI progressing to MII *in vitro* (25% compared to 62.2%, respectively). It is less than half when compared to the fertilization rate of retrieved sibling MII oocytes (69.5%). A high rate of multinucleated oocytes is also found in fertilized MI oocytes injected immediately after denudation [148].

In cases of poor responders and in patients with an unsynchronized cohort of follicles, where the presence of immature oocytes is frequent after stimulation [149], the use of immature oocytes is important in order to increase the number of embryos obtained in each cycle. Based on the assumption that oocyte maturity is a pre-requisite for obtaining normal fertilization, attempts have been made to mature GV and MI oocytes *in vitro* [147]. Despite the use of varying culture techniques and different stimulation protocols, such IVM oocytes consistently have lower fertilization rates, frequent cleavage blocks and overall retarded cleavage rate compared with sibling MII oocytes [147, 150]. The limited number of transfer cycles makes it difficult to draw solid conclusions about the value of transferring these embryos. It should be noted that the immature oocytes collected in stimulated cycles have already been under stimulation with high doses of gonadotropins and are exposed to hCG before retrieval. The nuclear maturation, cytoplasmic maturation and ensuing developmental capacity of these oocytes may very well be different in comparison with immature oocytes collected from small antral follicles of unstimulated ovaries in the typical IVM procedure [151].

Intracytoplasmic Sperm Injection – Factors Affecting Fertilization 133

rate [161]. In human oocytes, the cytoplasmic granularity can be homogeneous affecting the whole cytoplasm, or concentrated in the centre with a clear peripheral ring giving a darkened appearance to the cytoplasm [162]. The abnormal changes in the cytoplasm of MII oocytes may be a reflection of delayed cytoplasmic maturation that is unsynchronized with

**Figure 5.** Oocytes in first row represent different degrees of vacuoles in cytoplasm. Each oocytes in

Normal fertilization, embryo development and live birth are possible after ICSI in oocytes with thick zonae, abnormal morphology or repeated polyspermia following conventional IVF. The oocytes with extreme morphological abnormalities should not be discarded as ICSI can result in fertilization, cleavage and normal embryonic development [164, 161]. The zonafree oocytes may be fertilized normally after ICSI and develop to the blastocyst stage [165]. Pregnancy in human [166] and live birth in mouse [167] and pig [168] have been obtained

Repeated ICSI treatment can be useful or necessary because there is a high possibility of achieving normal fertilization if a reasonable number of oocytes with normal morphology are available and motile sperm can be found. If there are no motile sperm present in the first ejaculate, a second sample should be required followed by PESA or TESE to obtain motile sperm. In this way, a sufficient number of motile sperm for ICSI are usually found in most

A history of failed fertilization may be related to some gamete abnormality that may be modified or corrected at the next cycle. It has been documented that for a particular patient, fertilization results can be quite varied when followed through several ICSI cycles at the same centre [169]. The differences between fertilization rates are unexplained, although

nuclear maturity [163].

second row has increased central granularity.

after transfer of embryos resulting from zona-free oocytes.

**16. ICSI after previous ICSI cycle failure** 

men with severe asthenozoospermia.
