Does Physiological Intracytoplasmic Sperm Injection Improve Outcome in Men with Abnormal Semen Parameters: A Retrospective Cohort Study

The overall rising incidence of infertility has a significant contribution by the declining male factors.[ 1 ] Female factors contributing to poor outcomes in assisted reproductive technique (ART) cycles are well known and studied, but the male factors lack the same emphasis, even though it contributes to nearly half of the cases, either directly or in combination with female factors.[ 1 ] Multiple ways have developed to optimise outcome in male infertility; amongst those, most commonly used is the conventional intracytoplasmic sperm injection (ICSI) in which the best sperm, as assessed under the inverted microscope according to its motility and morphology by an embryologist, is injected to the oocyte.[ 2 ] However, the quality of sperms in terms of maturation and DNA integrity has always remained a question of concern in conventional ICSI. These have a strong association with poor embryo quality and increased miscarriage rates.[ 3 ] Many techniques have been developed in recent years to select the best sperm hoping to exclude these subtle flaws and positively impact the overall outcome of ART cycles. Of these methods, one is hyaluronic acid (HA) binding sperm selection for ICSI also called as physiological intracytoplasmic sperm injection (PICSI). The hypothesis behind HA-ICSI/PICSI method is that only the structurally and genetically mature sperm with good DNA integrity possess a hyaluronic receptor which is able to assimilate and dissolve the HA, the major component of cumulus oophorus to fertilise oocyte.[ 4 ] There are two methods available for sperm selection using HA binding. The first is solid-state platform in which dishes are conventional plastic culture dishes pre-prepared with three microdots of powdered HA, and the second is liquid-state platform containing viscous medium called as sperm slow (trade mark).[ 5 ] In the past, there have been inconsistencies in the outcome of studies involving PICSI. This could be mainly attributed to the heterogeneity amongst the various studies and the study population involved in them. The various studies conducted comparing conventional ICSI with PICSI in population with previous in vitro fertilisation (IVF) failure, sibling oocyte and teratozoospermic sperm have shown improved fertilisation, cleavage, blastulation rate along with good-quality embryo and implantation rate in the PICSI group.[ 6 7 8 ] However, a study conducted by Majumdar and Majumdar in cases of unexplained infertility showed no improvement in fertilisation rate or the quality of embryo but had been shown to reduce pregnancy losses in the PICSI group.[ 9 ] Cochrane review after analysis of randomised control trials had concluded that sperm selected by PICSI may have little or no effect on live birth or clinical pregnancy but may reduce miscarriage.[ 10 ] The aim of the present study is to compare the embryological and clinical outcomes between PICSI and conventional ICSI in men with abnormal semen parameters. We have analysed and compared the blastulation rate between the conventional ICSI and PICSI as our primary objective which is not so extensively studied in the literature (but would act as a more applicable parameter in clinics who vitrify/transfer both D3 and D5 embryos). Fertilisation, cleavage, clinical pregnancy and miscarriage rate were computed as the secondary outcomes.

The demographic profile of study and control groups, as shown in Table 2 , for age, body mass index and infertility duration was comparable. The clinical and other laboratory parameters were comparable, as shown in Table 3 , in both the groups, except for the total motile concentration of sperm which was higher in the study group (PICSI vs. conventional ICSI, 44 ± 21.3 vs. 32.2 ± 21.03, P = 0.005) while sperms with normal morphology (1.4 ± 0.8 vs. 1.8 ± 0.79, P = 0.01) were higher in the control group. Demographic profile BMI=Body mass index, SD=Standard deviation, ICSI=Intracytoplasmic sperm injection, PICSI=Physiological ICSI Laboratory and embryological parameters ***Total sperm concentration (million/mL) × volume (mL) × total motility percentage/100%. SD=Standard deviation, ICSI=Intracytoplasmic sperm injection, PICSI=Physiological ICSI, FSH=Follicle-stimulating hormone, AMH=Anti-üllerian hormone, TMSC=Total motile sperm count The fresh embryo transfer was done in 7/51 (13.7%) and frozen embryo transfer was done in 44/51 (86.3%) patients in the PICSI group, while in conventional ICSI, it was done in 11/54 (21.5%) and 43/54 (78.5%) patients, respectively. Amongst the embryological parameters, the mean number of fertilised oocytes, good-quality cleaved embryo, the embryos left for the blastocyst culture and the total number of blastocysts were comparable in both the groups [ Table 3 ]. The blastulation rate was significantly higher for the PICSI group (43.7%) as compared to the conventional ICSI (34.2%) ( P = 0.022) [ Table 4 ]. We also found that PICSI produced 42% more blastocysts as compared to ICSI (adjusted IRR = 1.423, 95% confidence interval = 1.014–1.998, P = 0.042) after adjusting for number of oocytes retrieved, age of both the partners, total motile sperm count and percentage of normal sperms according to morphology [ Table 5 ]. However, the fertilisation and cleavage rates between the groups showed no significant statistical difference (75.8% vs. 83.8%, 89.7% vs. 87.6, P > 0.05) [ Table 4 ]. Outcomes ICSI=Intracytoplasmic sperm injection, PICSI=Physiological ICSI Adjusted primary outcome IRR=Incidence rate ratio calculated by Poisson regression, CI=Confidence interval, ICSI=Intracytoplasmic sperm injection, PICSI=Physiological ICSI Statistically higher pregnancy rate (PICSI vs. conventional ICSI, 37.2% vs. 32%, P = 0.029) and lower miscarriage rate (5.3% vs. 11.7%, P = 0.005) were demonstrated in the PICSI group as compared to the ICSI group. The pregnancy rates for fresh embryo transfers, when considered individually, did not show any statistically significant difference (PICSI vs. conventional ICSI, 28.5% [2/7] vs. 27.2% [3/11], P = 0.079) between the groups. However, in frozen embryo transfers, the pregnancy rates were significantly higher for the PICSI group as compared to the ICSI group (38.6% [17/44] vs. 33.2% [14/42], P = 0.032) [ Table 4 ].

PICSI could be a promising tool in male factor infertility by increasing the number of good-quality blastocysts, clinical pregnancy rate and decreasing the miscarriage rate, though large randomised studies are needed for more affirm the evidence. SK: Concepts, design and definition of intellectual content and manuscript writing; SS: Concepts, investigation, manuscript writing, data collection and study design and data analysis; ASB: Design and definition of intellectual content; RG: Data collection and study design and data analysis; RA: Data analysis. Nil. SK is an associate editor with JHRS, but he was not involved in the peer review process or the editorial decision-making. The data are available with the corresponding author and willing to share it on request.

In our study, comparing PICSI with conventional ICSI, we found no difference in fertilisation and cleavage rate, but the adjusted blastulation rate was significantly higher in the PICSI group as compared to the control group of ICSI. There were also an associated higher implantation rate, higher clinical pregnancy rate and a reduced miscarriage rate in the PICSI group. The effect of subnormal semen parameters on the embryological parameters of assisted reproductive cycle is now well established in the literature.[ 14 ] However, despite the rampant use of conventional ICSI, which is being used to overcome issues caused by male factors, the optimal sperm selection method is yet to be discovered. The result of our study corresponds with the proposed theory of the sperm affecting the embryogenesis at two points: first, before the cleavage stage where it can affect both the fertilisation and cleavage rates, and second, after the cleavage stage where it can affect the blastulation rates and implantation rates of embryos along with the miscarriage rates. The later phase is dominated by the spermatic genome which can certainly and severely be affected by the sperm maturity, DNA fragmentation and other such factors, the effects of which can extend into the embryonic and clinical parameters.[ 15 ] The effects of high DNA fragmentation and its detrimental effect over embryonic phase and implantation rate are also demonstrated by Simon et al .[ 16 ] Hence, when HA bound sperm selection method is used, it generally selects a more genomic mature sperm with intact DNA and can improve the embryological outcome.[ 4 ] There are multiple studies conducted over a time on different groups such as previous ICSI failure, sibling oocyte and teratozoospermia which have shown results similar to our study of improved embryologic key performance indicators and good-quality blastulation rates with the PICSI group as compared to ICSI.[ 6 7 8 ] Although, in contrast to our study, a study conducted on 156 cases of unexplained infertility by Majumdar and Majumdar comparing PICSI with conventional ICSI for a similar outcome did not show any statistical difference.[ 9 ] Various underpowered studies conducted in male factor infertility and in unselected population have also shown increased pregnancy rate similar to our study.[ 17 18 ] A systemic review done by Beck-Fruchter et al ., which includes seven studies of heterogeneous group comparing PICSI and ICSI, has shown similar results as our study, namely better embryo quality and increased implantation rate, but no significant difference in other embryological parameters (cleavage and fertilisation rate) and clinical pregnancy rate with the use of PICSI.[ 4 ] Another systemic review by Avalos-Durán et al . which included two prospective studies done over the couples with male factor infertility only showed contrary results.[ 19 ] This was corroborated by a recent study conducted using sibling oocytes, which also showed no significant benefit of using PICSI over conventional ICSI.[ 20 ] A large randomised trial HBASelect published in 2019 comparing PICSI with conventional ICSI in infertile couples who underwent IVF for various indications has also shown a reduction in miscarriage rate which is similar to our study, though no effect on the live birth rate was noticed using PICSI. Even though live birth rate was the primary outcome of this trial, post hoc studies showed that one of the arms of this study involving older women showed an improved live birth rate using PICSI.[ 3 21 ] We are aware of the limitation of our study, majorly that is a retrospective study with small number of cases. Randomisation was not possible because of the retrospective nature. Other limitations include the fact that live birth rate and DNA fragmentation index were not factored into the study. This was because of commercial aspects associated with that of private practice. Although, in majority of the studies conducted over past, reduced miscarriage rate and increased implantation rate were seen, which is similar to the conclusion of our study.

A retrospective cohort study was conducted at our tertiary level assisted reproductive centre over the period of 9 months (April 2023 to December 2023) according to the Helsinki Declaration after the ethical approval taken from the organisation for informed waiver consent and anonymised usage of the participant data. Our inclusion criteria were the use of fresh semen having any abnormal semen parameters as per the WHO reference range (any abnormality in either one or more parameters amongst count, motility or morphology is considered), non-donor gametes cycle, fresh oocyte and sperm and the use of antagonist protocol for controlled ovarian stimulation.[ 11 ] Exclusion criteria were cryptozoospermia, globozoospermia, grossly abnormal oocyte (irregularities in shape and size [big and small], smooth endoplasmic reticulum, refractive bodies, central granulation, thick zona, debris and inclusions in perivitelline space), uterine factor (type 0 to type 4 fibroids, fibroids >5 cm and adenomyosis) and very poor ovarian reserve (anti-Müllerian hormone <0.3 ng/ml and surgical retrieval of sperm). This was done to maintain the homogeneity in the parameters which might influence outcomes. Over the duration of 9 months amongst 396 ART cycles, we identified a total of 164 patients having abnormal semen parameters, and amongst these, 104 couples fulfilled the enrolment criteria and were recruited for our study. The enrolled population were divided into two groups; study population ( n = 51) amongst those PICSI was used as a sperm selection method and controls ( n = 53) in which conventional ICSI was used. The primary outcome of the study was the Adjusted blastulation rate. This was defined as number of developed blastocysts (both day 5 and day 6- expansion of 3-6; inner cell mass and trophoectoderm of Grade A/B as suggested by Gardner and Schoolcraft) per all embryos that were left to culture up to blastocyst-stage (it excludes Day 3 embryo which were transferred or frozen) [ Table 1 ].[ 12 ] The decision for transfer/freezing and extended culture for the day 3 embryo was taken after the discussion between patient and the clinicians as per the clinical profile of patient, number and grade of embryo. Definition of outcomes *Grade 1 and Grade 2 of cleaved (Day 3 embryo), **Expansion 3–6. ICM/TE Grade A/B. ICM=Inner cell mass, TE=Trophoectoderm, ICSI=Intracytoplasmic sperm injection The secondary outcomes were taken as fertilisation rate (defined as the ratio between the number of fertilised oocytes 2 pro-nuclei [2PN] and the number of metaphase II [MII] oocytes), good-quality cleaved embryo formation rate (the ratio between the total number of cleaved embryos and the total number of fertilised oocytes [2PN]), clinical pregnancy rate (one or multiple gestational sacs seen by ultrasonography (USG) of all the embryo transfer done) and miscarriage rate (defined as number of loss of pregnancy before 24 weeks of the total number of pregnancies) [ Table 1 ]. For the cleavage stage embryos, we considered grades 1 and 2 of embryo as per the Istanbul consensus.[ 13 ] Semen was collected after 2–5 days of abstinence by masturbation on the day of oocyte retrieval. It was then kept for about 30 min for liquefaction. Processed semen sample was then assessed for count, motility and morphology.[ 11 ] Total motile sperm count was calculated for each patient (calculated by multiplying the concentration of sperm (million/ml) by volume (ml) and progressive motility divided by 100 percentages). To extract motile sperm from seminal plasma and debris, density gradient centrifugation or swim-up methods were used. Density gradient sperm preparation involved layering semen over columns of different density gradient media in a centrifuge tube (1:1:1 ratio). The sample was centrifuged at high speed (typically 300–500 ×g for 15–20 min) and then desired sperms collected from the pellet, rewashed with fresh media yielding a sample enriched with high-quality sperm for further procedure. In swim-up technique, the processed sample was layered with a small amount of fresh 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or culture media (0.2–0.5 µl) and kept in the incubator accordingly for 10–15 min. Sperms that swam up to the surface of the media were collected and separated in another fresh tube for further use. We used pre-coated PICSI dish with HA, ready-to-use solid-phase PICSI dishes, as per availability at our centre (PICSI® Sperm Selection Device, Cooper Surgical) [ Figure 1 ]. Physiological intracytoplasmic sperm injection dish We used an appropriate medium (such as human tubal fluid) to hydrate the PICSI plate covered with HA. As directed by the manufacturer, we incubated the dish for the pre-determined amount of time (10–15 min generally) at 37°C, 5% CO 2 and high humidity. A tiny amount (about 5–10 µL) of the processed sperm solution was applied to HA-coated surface of the PICSI dish. We left the sperm 10–15 min to incubate on the dish. Spermatozoa that are fully developed and capable of fertilisation were attached to the HA during this period. Then, we located and chose the sperm attached to the HA with an inverted microscope along with assessing the morphology simultaneously. For controlled ovarian stimulation, conventional antagonist protocol was used and ovum pick-up was done at 35 h post-trigger. The dosages of gonadotropins, start of the antagonist and determination of trigger were individualised as per patient parameters. After collection of oocyte, mature (MII stage) oocytes that are ready for injection were collected after eliminating cumulus cells by a mixture of physical and chemical denudation. We injected the chosen HA-bound sperm into each mature oocyte’s cytoplasm using a micromanipulator. Culture: After injection, we put the oocytes in an appropriate culture medium. Provide the ideal environment for fertilisation and the subsequent development of the embryo. The culture media was not changed throughout the entire study period. Following injection of sperm, the fertilisation rate was assessed after 17–18 h of injection, cleavage rate (after 66–74 h) and blastulation rate (after 118–144 h). Those cycles which are planned for fresh embryo transfer underwent the procedure either as day 3 (cleavage) or day 5/6 embryos (blast). Rest embryos were frozen for frozen embryo transfer later as per the patient individual profile. Following embryo transfer patients were advised for beta-human chorionic gonadotropin (HCG) test to detect pregnancy after 2 weeks of embryo transfer. Those who had positive beta-HCG report (>25–50 mIU/ml and 66% or more rise in 48 h) were asked to report for USG after 2 weeks of test to confirm pregnancy. Clinical pregnancy was defined as gestational sac seen by USG. Any unfavourable events in terms of miscarriage rate (loss of pregnancy) before 24 weeks of pregnancy were recorded. Continuous data were first checked for normality by visual inspection of histogram and quantile plot and statistically by Kolmogorov–Smirnov test. Normally distributed data were reported as mean (standard deviation [SD]), and non-normally distributed data were reported as median (interquartile range). The outcome measures were calculated for the study group and the control group and were compared using independent Student’s t -test. Categorical variables were analysed using either the Chi-square test or Fisher’s exact test. Continuous variables are presented as mean ± SD, and categorical variables are presented as absolute number and percentage. For estimating the association of primary outcome (number of blastocysts at days 5–6) with clinically important covariates, Poisson regression was done as the outcome was count variable after check for dispersion. Bivariable model was estimated first, followed by multivariable model. Association was reported as incidence rate ratio (IRR) of blastulation between PICSI and ICSI. For all analyses, P < 0.5 was considered statistically significant. Statistical analyses were performed using SPSS statistical software (version 25.0, Chicago, IL, USA).