Influence of sperm morphology in ICSI cycle outcomes: a retrospective study

INTRODUCTION

Assisted reproductive techniques (ARTs) have been used with increased frequency since the birth of first in vitro fertilization (IVF) baby in 1978, and are the treatment of choice in indicated cases of male and female infertility.^[1] Infertility is defined as the failure to accomplish a clinical pregnancy after 1 year or more of regular intercourse as per WHO. Approximately 10% to 15% couples all over the world suffer from infertility, of which 25% to 30% are due to male factors, 20% to 35% are due to female factors, and 20% are unexplained.

Intracytoplasmic sperm injection (ICSI) is an IVF procedure in which a single sperm cell is injected directly into the cytoplasm of an egg to form an embryo which is then transferred into the maternal uterus for obtaining a pregnancy.

The ICSI was first introduced as an add-on IVF procedure in 1992, almost 22 years after the birth of the first IVF baby. Previously ICSI was used for severe male factor infertility and those with unexplained infertility but now has been widely accepted for several other indications such as previous IVF failure.

The ICSI involves sperm selection of live sperms based on their morphologic features observed under an inverted microscope, at an optical magnification of 400×. At this magnification, some of the morphologic anomalies can be detected such as number of tails or other tail and neck abnormalities, or the presence of cytoplasmic droplet. The advantage of this method of sperm selection is that no staining of the sample is involved and thus viability is not affected. However, only major morphologic abnormalities can be detected by this method.

Semen analysis is commonly used in the evaluation of the male partner among infertile couples.^[2] Of the various semen analysis parameters, strict Sperm morphology assessment as described by Kruger has been proposed to be one of the most informative in differentiating between fertile and infertile men. It has been considered a biomarker of sperm fertilizing capacity, ICSI is an IVF procedure in which a single morphologically selected sperm cell is injected directly into the cytoplasm of an egg to form an embryo.

Severe abnormalities in sperm morphology, as in the case of teratozoospermia, are associated with poor fertilization during IVF cycles. In such clinical scenarios, ICSI results in higher fertilization rates than conventional IVF, without affecting embryo quality.^[3]

The present study was conducted to evaluate whether the sperm morphology has any influence on the ART outcome in ICSI cycles.

MATERIALS AND METHODS

All patients satisfying the inclusion and exclusion criteria were recruited into the study.

Data from 1000 ART cycles of couples who had undergone IVF ICSI cycle at Southend Fertility and IVF between January 2016 and January 2021 were analyzed in this retrospective study.

Data included information on the process of IVF-ICSI such as controlled ovarian stimulation, cycle monitoring, oocyte recovery, insemination or injection of oocytes, fertilization check, and culture of embryos to cleavage or blastocyst stage and finally embryo transfer.

At the time of oocyte retrieval, semen was analyzed for count, motility, and morphology. It was then prepared for use in ICSI using the double density gradient method of sperm separation.

A routine semen analysis was performed according to World Health Organization guidelines (WHO, 2010) using a Makler chamber.

Slides were prepared for sperm morphology assessment using the Diff-Quik method and then assessed for morphology. Sperm morphology grading was carried out according to the Tygerberg criteria.^[1,9]

A sperm was considered morphologically normal if the head was normal (normal shape, normal size, having an acrosome, and lacking midpiece or tail defects). Assessment was made by using strict Kruger criteria, to the extent possible given the limited magnification.

The main defects recorded in morphologically abnormal sperms were elongated or tapered head (at least twice smaller in width compared to normal), amorphous head, broken neck (length axis of the head deviated at least 30° from the midpiece axis owing to a breakpoint between the head and midpiece), and presence of a cytoplasmic droplet (regardless of the size of the droplet relative to the head size).

After the oocyte retrieval, the oocytes were incubated for 2 hours and then denuded to remove the surrounding cumulus in a hyaluronidase-containing medium and then rinsed in HEPES-buffered IVF medium. Five droplets of culture medium, each 5 μL, were placed around one droplet in the center of a plastic culture dish. These were covered immediately with sterile, equilibrated paraffin oil. The center droplet was subsequently replaced by a droplet of polyvinyl pyrrolidone (PVP) solution. Fresh semen sample was taken and prepared. A 1 μL aliquot of the sperm suspension was added to the middle of the PVP droplet. Only mature oocytes having a polar body in the perivitelline space were placed in the media droplets. Sperm morphology was evaluated under the inverted microscope. Selected spermatozoon was immobilized and aspirated into the ICSI pipette and oocyte was inseminated. After injection, the oocytes were transferred to culture dishes containing cleavage medium. Oocytes were then examined 16 to 18 hours after ICSI to assess for fertilization, that is, the presence of two distinct pronuclei and two clear polar bodies. Serial evaluation of embryo morphology was performed as per Istanbul 2010 consensus.^[4] The highest morphologically scored embryos were selected for transfer to the patient on the day 2 or 3 or 5. Extra embryos, if available, were cryopreserved by vitrification using Kitazato medium. Pregnancy was detected by measuring serum beta human chorionic gonadotropin levels.

RESULTS

A total of 1000 ART-ICSI cycles were included in the analysis, of which 600 cycles belonged to group A (poor prognosis cases) and 400 cycles to group B (good prognosis control). The baseline characteristics, such as age, type of infertility, and semen parameters such as volume, count, motility, debris, agglutinations, grade A motility, grade B motility, total progressive (A+B) motility, morphology assessments, and type of protocol used for stimulation, were comparable between the two groups.

Table 1 compares the semen parameters between the two groups. It shows statistically significant difference in motility and morphology in the two groups (P = 0.001). Table 2 compares the outcome parameters between the two groups.

Table 1 Male factor category and semen parameters

Table 2 Outcome parameters between the two groups

Table 3 compares the baseline characters in the female partner in the two groups. Among all the baseline characters, significant difference was reported in the age between the two groups (P = 0.005).

Table 3 Baseline female parameters

In the 1000 patients analyzed, a total of 6871 oocytes were injected at ICSI, of which 4049 oocytes belonged to group A and 2822 oocytes belonged to group B. Of the 6871 oocytes injected, 6275 got fertilized, giving a fertilization rate of 91.3%. On comparing the fertilization rate of the two groups, a fertilization rate of 90.1% (3650/4049) was reported in group A and 93.01% (2625/2822) was reported in group B, which was statistically different (P < 0.001).

The cleavage rates in groups A and B, respectively, were 91% and 89%. In groups A and B, grade A embryo development rates were 51% and 48%, grade B embryo development rates were 30% and 28%, and grade C embryo development rates were 11% and 13%, respectively.

Of the secondary outcome, in fresh embryo transfer cycles, pregnancy rates of 60% in group A and 62% in group B were observed, and a pregnancy rates of 40% in group A and 41% in group B were obtained in frozen embryo transfer cycles. However, the pregnancy rates in the two groups were not found to be statistically significant in both fresh and frozen embryo transfer cycles.

It was also observed that injection of morphologically abnormal spermatozoa did not influence the ICSI survival rate, which was similar for the different types of morphologically abnormal spermatozoa and for morphologically normal spermatozoa (overall, 93% of injected oocytes). Once fertilization occurred, embryo quality was good for all types of abnormal spermatozoa (grade A, good- to excellent-quality embryos), except for spermatozoa with broken necks, with which only good to excellent quality, or poor quality grade B and group C embryos were obtained.

DISCUSSION

In the 1000 ICSI cycles analyzed, we studied the effect of morphologically normal sperm or abnormal sperm cells on ART cycle outcomes. Selection of the “most normal-looking” spermatozoon as observed under the inverted microscope for microinjection is a very important selection step within the procedure. More normal-looking spermatozoa are injected than expected from the Kruger morphology assessment on the semen sample. This study compared ICSI outcomes in couples with normal morphology infertility and male infertility with teratozoospermia to determine if having sperm morphology of less than 4% or greater than 4% were related to the ICSI outcomes.

In the present study, the spermatozoa concentration, total motility, progressive motility(A+B), and other morphology parameters within the two groups were also compared and a statistically significant difference was observed between the two groups in terms of concentration, motility, and morphology parameters, whereas no difference was observed in sperm volume.

Among the primary outcome measures, a statistically significant difference was reported in the fertilization rate, cleavage rate, and embryo development rate between the two groups. However, there was no difference in the pregnancy rates between the two groups. We found in this retrospective study that there is no such difference in fresh embryo transfer pregnancy rates in group A (40%) and group B (41%). On the other hand, in frozen embryo transfer, pregnancy rates in groups A and B were 60% and 62%, respectively, but statically not significant. However, we also observed that frozen embryo transfer pregnancy rate was better than fresh embryo transfer pregnancy rate and this indicates that though morphology affects the embryo development, it does not have any adverse implication on the ART outcome.

As per literature, fertilization failure may be due to sperm factors which have been related to sperm morphology, sperm nuclear morphology, acrosomal defects, and sperm chromatin status. However, sperm morphology may not be a critical factor for fertilization using ICSI because other processes such as failure of oocyte activation may be involved in this adverse result also.

As given in literature, in our study also we found a higher pregnancy rate after frozen embryo transfer when compared with a fresh embryo transfer. However, the pregnancy rates in the two study groups were not significantly different in either fresh or frozen embryo transfer cycles. This suggests that the morphology of sperms is not related to the success rates of ICSI.

Our results were comparable to the observations of Preetha et al.^[5] They conducted a retrospective study to understand the influence of spermatozoa morphology on ICSI cycle outcome parameters in couples with male factor infertility, and found no significant relation of sperm morphology with ICSI outcome parameters, such as fertilization rate, embryo development rate, embryo quality rate, pregnancy rate, miscarriage rate, and live birth rate. Various other studies are also in agreement with our observations that there is no correlation of sperm morphology with ART-ICSI outcomes.^[6,7,8,9] This also suggests that the Kruger strict morphology criterion for fresh semen sample does not correlate with ART-ICSI outcomes, and the microscopic examination and selection of spermatozoa under the inverted microscope during ICSI can yield similar results in good and poor morphology groups.

In another retrospective study, Palermo et al.^[10] evaluated the assessment of sperm morphology based on “strict morphologic criteria”^[11,12,13] and discriminated three categories in relation to the predicted outcome of standard ART treatment: excellent (>14% morphologically normal spermatozoa), good (5–14%), and poor prognosis (<5%).^[7] They reported that none of the sperm parameters correlated with the outcome of ART. This further suggests that sperm morphology does not affect ART outcome.

In ART-ICSI cycles, earlier studies have shown that semen samples with poor Kruger morphology have similar fertilization and pregnancy rates to those with normal morphology.^[14] Individual sperm morphology assessed at the moment of ICSI correlated well with fertilization outcome, but did not affect embryo development.^[15] The implantation rate was lower when only embryos resulting from injection of an abnormal spermatozoon were available. Literature is contradictory with respect to strict sperm morphology and its effect on ICSI cycle implantation rate.

This was similar to our findings that there was a significant difference in the fertilization rate among the two study groups. However, in our study, no difference was found in the implantation rate between the two groups.

Overall, the findings of the other retrospective studies conducted by Kihaile et al. are consistent with the studies by McKenzie et al.^[16] and French et al., which show equivalent outcomes of ICSI cycles for strict sperm morphology of 0% versus morphology of ≥1%. The ability of ICSI to achieve normal fertilization independent of sperm morphology can be explained by the presence of sperm-borne oocyte activating factor.^[17,18]

According to another retrospective study conducted by Svalander et al.^[19] sperm morphology may not be a critical factor for fertilization using ICSI because many natural processes, such as the penetration of the zona pellucida, are bypassed and no correlation was observed between sperm morphology and fertilization rate after ICSI.

However, it is difficult to correlate ICSI to morphology of spermatozoa. ICSI involves the selection of one sperm and injected into cytoplasm of oocytes; all the sperm abnormalities are there by bypassed by using ICSI procedure. In our study also, we did not find any effect of sperm morphology on ICSI cycle outcomes.

This study has certain limitations, including the retrospective nature of the analysis. Also, there may be some female factors which were not diagnosed in the two groups. Moreover, couples who were diagnosed with infertility had more than one factor involved. Apart from the male factor, the female may also be diagnosed with PCOS, advanced maternal age (AMA), poor ovarian reserve (POR), decreased ovarian reserve (DOR), thyroid, endometriosis, tubal factors, and many unknown factors which also affect ART outcomes. Patients’ data were collected with either having normal morphology or abnormal morphology criteria, according to Kruger criteria. In this study, most important criterion is to see male sperm morphology effects on the ICSI outcomes. The low magnification and low resolution of the sperm morphology assessment on motile spermatozoa before ICSI is a limitation of our study. Studies assessing sperm morphology of the inseminated spermatozoa during ICSI using special imaging systems suggest that sperm morphology shows a significant and high correlation with fertilization and pregnancy rate. Newer techniques for sperm selection such as intracytoplasmic morphologically selected sperm injection (IMSI) and motile sperm organellar morphology examination (MSOME) have been introduced. This concept has been taken to the subcellular level in new techniques, such as IMSI and MSOME. Larger studies using this technology may provide stronger correlation of sperm morphology with ART-ICSI outcomes.

CONCLUSION

Our study finding concluded that the Kruger strict morphology criterion for sperm morphology does not correlate with ART-ICSI outcomes in male factor infertility. The limitation of this study is its retrospective nature and a prospective randomized study can bring more light to the present knowledge on this aspect. A larger sample size and having genetic information are needed to draw a proper conclusion. Further, larger prospective trials evaluating the influence of sperm morphology after processing with density gradient centrifugation on ICSI cycle may help prognosticate ART cycle outcomes.