Sperm vacuoles negatively affect outcomes in intracytoplasmic morphologically selected sperm injection in terms of pregnancy, implantation, and live-birth rates

Ermanno Greco, M.D., Filomena Scarselli, M.Sc., Gemma Fabozzi, M.Sc., Alessandro Colasante, Ph.D., Daniela Zavaglia, M.D., Erminia Alviggi, Ph.D., Katarzyna Litwicka, M.D., Maria Teresa Varricchio, M.D., Maria Giulia Minasi, M.Sc., and Jan Tesarik, M.D.

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The importance of sperm morphology in achieving a successful in vitro–intracytoplasmic sperm injection (IVF-ICSI) outcome has represented one of the most controversial issues of the last two decades. Initially, no correlation between the rate of morphologically normal spermatozoa and ICSI outcome was reported in the literature (1–3). However, to determine whether subtle sperm morphologic characteristics affect ICSI outcome and to identify those that are more relevant, Bartoov et al. (4) developed the method of motile sperm organellar morphology examination (MSOME). By examining sperm morphology at high magnification (>6,600), these investigators demonstrated that spermatozoa without morphologic anomalies give higher fertilization and pregnancy outcomes. After additional studies confirmed the same evidence (5–7), the procedure of including the MSOME selection method together with a micromanipulation system named intracytoplasmic morphologically selected sperm injection (IMSI) has been gradually introduced in the world of assisted reproduction as an alternative to conventional ICSI. In the literature, various studies have compared ICSI and IMSI, and controversial conclusions have been drawn, especially concerning fertilization and top-quality embryo rates (8–12). Some studies have shown that IMSI improves reproductive outcomes in cases of male factor infertility and/ or previous failed ICSI attempts in terms of implantation and clinical pregnancy rates as compared with conventional ICSI (10, 13, 14).

On the other hand, IMSI and conventional ICSI seemed to provide comparable laboratory and clinical results when an unselected infertile population was evaluated (11). The first studies concerning the importance of high magnification sperm selection were focused on the observation of the following elements: acrosome, postacrosomal lamina, neck, mitochondria, tail, and nucleus. The main parameters to be analyzed were the shape and size of the nucleus (length and width) and the presence/absence of vacuoles. More recently, the analysis of the presence of vacuoles has gained particular prominence with respect to the outcomes of assisted reproductive technology (ART). First, Berkovitz et al. (15) reported a negative correlation between the size of nuclear vacuoles and chromatin stability. Then, additional studies confirmed the relationship between the presence of large nuclear vacuoles and the impairment of sperm chromatin condensation (16, 17), a process that has been demonstrated to exert a critical role in embryonic development in protecting the paternal genome before fertilization (18). The influence of nuclear vacuoles in embryo development has been already demonstrated in various species by studies that have shown that early embryonic death and reduced fertility occur when oocytes are fertilized by vacuolated spermatozoa (19, 20).

Vanderzwalmen et al. (21) were the first group to show that the presence of vacuoles has an impact on human embryo competence, affecting development up to the blastocyst stage and being related to diminished pregnancy and implantation rates. Other studies have supported similar findings and shown that IMSI improves fertilization and blastocyst formation rates (22, 23). However, only a few surveys have focused on the effect of vacuolization pattern on the clinical outcomes of ART cycles (13, 15, 24). Our study evaluated the influence of the presence of sperm vacuoles on IMSI clinical outcomes, including live-birth rates.

MATERIALS AND METHODS

Patients

A total of 101 couples with at least two failed ICSI attempts and impaired sperm morphology [percentage of normally conformed spermatozoa %3% according to Kruger strict criteria (25)] were retrospectively included in this study. The study group included women younger than 38 years old (mean age standard deviation [SD] 36.15 3.93) with basal follicle-stimulation hormone (FSH) < 10 IU/L (mean value SD 7.28 5.47), body mass index [BMI ¼ weight (kg)/height (m2 )] <27 (mean value 22.45 5.47), menstrual cycle range 24–35 days (intraindividual variability 3 days), and normal karyotype of both subjects (Table 1). Patients in which the semen sample was derived from a cryopreserved sample, from testicular biopsies or with disorders such as polycystic ovary syndrome (PCOS), pelvic endometriosis, metabolic and quality autoimmune syndromes, which could affect oocyte competence, were excluded from the study. The couples were also evaluated on the basis of infertility factors: male factor infertility where there is a moderate or severe oligoasthenozoospermia according to World Health Organization criteria (26), tubal factor infertility where the woman had a hysterosalpingography or laparoscopy diagnosis of tubal infertility, and idiopathic factor infertility where the infertility cannot be explained.

Ovarian Stimulation

Ovarian stimulation was conducted in all patients, as described elsewhere (27), using a gonadotropin-releasing hormone (GnRH) agonist (buserelin acetate, 0.2 mg, twice daily; Suprefact; Aventis Pharma) started on day 21 of the menstrual cycle, recombinant FSH (Puregon 100 IU; Organon), and human chorionic gonadotropin (hCG, Gonasi; Amsa). Ovulation was induced with 10,000 IU of hCG (Gonasi; Amsa) when two to three follicles of 18–20 mm diameter were observed by ultrasound examination, and blood 17bestradiol levels exceeded 1,000 pg/mL. Oocyte retrieval was performed 36 hours after hCG administration under transvaginal ultrasound-guided puncture of the follicles.

Groups

Patients were divided into two groups according to sperm morphology and vacuolization pattern. Group A included patients whose spermatozoa retrieved for injection were classified as type I or II (good quality spermatozoa). Group B included patients in which spermatozoa retrieved for injection were classified as type III or IV (low quality spermatozoa).

IMSI Procedure

IMSI Procedure

Sperm samples evaluation and preparation. Sperm samples were collected by masturbation after 3 to 5 days of sexual abstinence and examined by microscopy at 40 magnification after liquefaction. Sperm concentration was assessed using a Makler counting chamber, motility was assessed according to World Health Organization criteria (26), and morphology according to Kruger’s strict criteria (25). All sperm samples were prepared by the swim-up technique immediately after oocyte retrieval. The whole semen sample was washed by centrifugation at 400 g for 10 minutes in HEPES-buffered medium supplemented with human serum albumin (Qunn’s Sperm Washing Medium; Sage). Then, 0.3 mL of medium (Quinn’s Advantage Fertilization HTF Universal Medium; Sage) was gently layered over the resuspended pellet and the sample was incubated in an atmosphere of 37C and 6% CO2 for 30 minutes. Finally, the upper layer containing motile sperm was removed and placed in a new tube and maintained in an atmosphere of 37C and 6% CO2 until IMSI was performed. Preparation for sperm retrieval and immobilization. A glassbottom dish (GWSt-5040; WillCo Wells) covered with mineral oil was prepared with two 5-mL microdroplets (md-1 and md- 2) of 7% polyvinylpyrrolidone solution (PVP 7% Solution; Sage), in one of which (md-1) a 2-mL aliquote of sperm cell suspension was transferred. Spermatozoa were selected at 12,500 magnification in an inverted microscope (AM6000; Leica) equipped with Normarski differential interference contrast optics, HCX PL FLUOTAR 100/1.30 oil objective lens, and variable zoom lens. The inverted microscope had a 37C heated stage, two Narishige electric coarse movement controls, two hydraulic micromanipulators, and two Narishige injectors for holding the oocyte and the injector pipette to retrieve and inject spermatozoa during IMSI (Narishige Ltd.). Spermatozoa were selected in md-1, then they were moved into md-2 and immobilized by crushing their tails. Finally, a picture of every spermatozoon was taken before injection. Criteria for selecting spermatozoa suitable for injection. Spermatozoa were classified according to their vacuolization pattern (type I to IV) in a way similar to that described by Vanderzwalmen et al. (21) (Fig. 1): Type I: normal head shape and absence of vacuoles or one small vacuole (<4% of the head volume) (Fig. 1A). Type II: normal head shape and presence of maximum two small vacuoles (one <4% and one >4% of the head volume) (Fig. 1B). Type III: normal head shape and presence of three or more small vacuoles (the combined total volume of all vacuoles have to be <4% of the head volume) (Fig. 1C). Type IV: abnormal head shape in conjunction with the presence of large or small vacuoles (Fig. 1D). Type I and II are good quality spermatozoa, while type III and IV are low quality spermatozoa (abnormal vacuolization). Sperm selection was undertaken at 38 hours after hCG administration. Type III and IV spermatozoa were chosen only when type I and II spermatozoa were not found after at least 50 minutes of search.

Microinjection

Once more suitable spermatozoa were selected as reported above, conventional microinjection under a microscope at 400 magnification was performed at 39 hours after hCG administration. Microinjection was performed in a plastic culture dish (cat. no. 1006, Falcon; Becton-Dickinson Labware) containing microdroplets (10 ml) of HEPES-buffered medium (Quinn’s Advantage Medium with HEPES; Sage) supplemented with 5% human serum albumin (Sage) arranged in two lines of three droplets, and one 10-mL microdroplet of 7% polyvinylpyrrolidone solution (PVP 7% Solution; Sage) in the center of the dish between the two lines of HEPES-buffered medium droplets. To prevent evaporation, mineral oil (Oil for Tissue Culture, SAGE, USA) was layered on the microdroplets in the injection dish. After at least 20 minutes of injection-dish incubation at 37C, selected spermatozoa were transferred into the PVP microdrop, and an oocyte was placed in each surrounding drop. A single spermatozoon was then aspirated in the injection pipette after a new process of tail crushing. The pipette containing the spermatozoa was then moved into a drop containing the oocyte, and sperm injection was performed as described elsewhere (28). The same inverted microscope used for sperm retrieval equipped with Hoffman Modulation contrast was used for sperm injection and assessment of oocyte quality, fertilization, and further development. The microtools used for sperm injection were commercial microtools (Origio Humagen pipets).

Oocyte Preparation and Embryo Culture

After retrieval, cumulus corona complexes were incubated in fertilization medium (Quinn’s Advantage Protein Plus Fertilization Medium; Sage) until denudation was performed. For all oocytes, denudation was obtained by brief incubation for 10 seconds in HEPES-buffered medium (Quinn’s Advantage Medium with HEPES; Sage) containing 20 IU/mL of hyaluronidase fraction VIII (80 IU/mL in HEPES-HTF; Sage). Subsequently, the oocytes were gently aspirated in and out of a plastic pipette (Flexipet, 170 and 140 mm i.d.; Cook Australia) to allow the complete removal of the cumulus and corona cells. Only oocytes with the first polar body extruded (metaphase II [MII]) were selected for sperm injection. Finally, injected oocytes were moved to the cleavage medium (Quinn’s Advantage Protein Plus Cleavage Medium; Sage).

Assessment of Fertilization and Zygote and Embryo Quality

Fertilization was assessed 16 to 18 hours after injection using the scoring system developed by Tesarik and Greco (29), and the fertilization rate was calculated as the ratio between fertilized oocytes and the number of injected oocytes. Embryo quality was evaluated two times: first,48 hours (day 2) after injection; then 1 hour before embryo transfer, approximately 72 hours after injection (day 3), using the scoring system reported elsewhere (28). The cleavage rate was defined as the total number of day-3 embryos divided by the total number of fertilized oocytes.

sperm vacuoles

Embryo Transfer, Luteal Phase Support, and Detection of Pregnancy

All embryo transfers were performed on day 3 with the use of a Soft-Pass Embryo Transfer Catheter (K-J-SP-681710; Cook Ireland Ltd.), and the number of transferred embryos depended on the age of the patient and on the embryo quality. Surplus embryos were cryopreserved. The luteal phase was supported as described elsewhere (27). The pregnancy test was considered positive when b-hCG reached values over 60 IU/L 13 days after embryo transfer. A clinical pregnancy was confirmed by observation of a fetal heartbeat at ultrasound analysis 7 weeks after the establishment of pregnancy. Pregnancy rate was defined as the number of positive b-hCG cases divided by the total number of embryo transfers. The clinical pregnancy rate was defined as the number of cycles with at least one fetal sac with heartbeat divided by the total number embryo transfers. The implantation rate was calculated as the number of fetal sacs with heartbeat divided by the number of transferred embryos, and the live-birth rate as the number of pregnancies with at least one delivered baby divided by the total number embryo transfers.

Ethics

This study was approved by the institutional ethics committee of European Hospital, and oral informed consent was obtained from all patients. All experimentation was performed according to the Helsinki declaration of 1975 and its modifications.

Statistics

Two-tailed Fisher’s exact test and Student’s t test were used to test the significance of the differences between the two groups. P<.05 was considered statistically significant.

Results

ICSI

A total of 101 consenting couples with a history of repeated ICSI failures and impaired sperm morphology were enrolled in IMSI cycles from January 2010 to December 2010. Biologic and clinical data were retrospectively analyzed, and the patients were divided into two groups, A and B, according to the morphology of the spermatozoa retrieved for injection. Group A included couples in which good quality spermatozoa (types I and II) were retrieved for injection (n ¼ 63 patients), and group B included couples in which only low quality spermatozoa (types III and IV) were available for injection (n ¼ 38 patients). No statistically significant differences were observed between groups A and B with regard to the population parameters (Table 1). In both groups, three patients did not have embryo transfer because oocyte fertilization did not occur. In group A, a total of 464 MII oocytes were injected with spermatozoa selected at high magnification, and 342 (73.7%) oocytes were observed to be fertilized 16 to 18 hours after injection. In group B, the number of MII oocytes injected with spermatozoa selected at high magnification were 251, and 168 (66.9%) oocytes were fertilized. No statistically significant differences between the two groups were observed with regard to the fertilization rate (73.7% vs. 66.9%), cleavage rate (89.5% vs. 85.7%), or the rate of top quality embryos (grades A þ B) (84.0% vs. 84.7%) (Table 2). Statistically significant differences in the percentage of pregnancy, clinical pregnancy, implantation and live-birth rate were detected between the two groups. A positive value of serum b-hCG concentration was recorded in 28 of 60 cycles in group A and in 8 of 35 cycles in group B, which meant a pregnancy rate of 46.7% and 22.9%, respectively (P<.05). The clinical pregnancy rate was 25 (41.7%) of 60 in group A and 6 (17.1%) of 35 in group B (P<.05). In group A, 34 embryos implanted out of 147 embryos replaced, and in group B, only 6 of 86 embryos transferred implanted, for an implantation rate of 23.1% versus 7.0%, respectively (P<.05). Finally, the live-birth rate was 22 (36.7%) of 60 in group A and 5 (14.3%) of 35 in group B (P<.05) (Table 3).

Discussion

The evidence that sperm morphology plays a key role in the achievement of a positive outcome in an ART cycle is increasingly gaining consensus in the scientific community. In consideration of this, more strict techniques for selecting morphologically normal spermatozoa have been developed recently. Intracytoplasmic morphologically selected sperm injection is a technique that allows a more accurate selection of normal sperm. It uses magnification of up to 6,000 times and reveals subtle morphologic anomalies that are difficult to detect at the 400 magnification used with conventional ICSI (4, 10). Since the introduction of IMSI, sperm vacuoles have become one of the most studied type of malformations, but no data are available in the literature concerning the impact of nuclear vacuoles on live-birth rates. Our retrospective study has demonstrated that the presence of sperm vacuoles significantly influences clinical outcomes of an ART cycle in terms of pregnancy, implantation, and live-birth rates. In the past, various investigators reported that neither the type nor the extent of sperm impairment was an important influence on the outcome of ICSI with regard to fertilization, embryo development, or pregnancy rate (1–3, 30). Our data support this evidence only in part—no statistically significant differences were observed between group A (patients with good quality spermatozoa) and group B (patients with low quality spermatozoa) with regard to fertilization rate (73.7% and 66.9%, respectively), cleavage rate (89.5% and 85.7%, respectively), or rate of top quality embryos (84.0% and 84.7%, respectively).

clinical outcomes

However, the pregnancy rates were statistically significantly different (46.7% and 22.9%, respectively). The same result was confirmed when other clinical outcomes were observed. A statistically significant difference between groups A and B was detected for the clinical pregnancy rate (41.7% vs. 17.1%, respectively), implantation rate (23.1% vs. 7.0%, respectively), and live-birth rate (36.7% vs. 14.3%, respectively). From our data, no statistically significant difference was found in ‘‘early’’ ART outcomes (fertilization rate and embryo quality) but statistically significant differences were detected in the ‘‘late’’ outcomes (pregnancy, implantation, and livebirth rates). Our hypothesis is that the presence of vacuoles and their size influence the outcome of an ART cycle by acting late during embryo development. Various studies have demonstrated that sperm-derived factors can influence preimplantation embryo development, a phenomenon referred to as the paternal effect (31–35). The paternal effect may manifest early at the zygote stage, causing delayed cleavage and increased embryo fragmentation (36), which is known as the early paternal effect. It also has been demonstrated that the paternal effect can negatively influence preimplantation embryo development and clinical outcomes in the absence of any detectable impairment in zygote development, cleaving speed, or embryo quality, which is referred to as the late paternal effect (34, 35). In light of our results, we speculate that vacuoles are an example of a sperm factor contributing to the late paternal effect. In fact, the analysis of our data showed no impairments at the zygote and/or early cleaving embryo stages in cases of the presence and/or increased extent of vacuoles, yet we observed a statistically significant difference in the clinical outcomes (pregnancy, implantation, and live-birth rates).

Because all embryos were transferred on day -3, we have no data concerning blastocyst development, but it has been reported in the literature that the presence of vacuoles already begins to affect embryo development at blastocyst stage (11). Various investigators have demonstrated an association between sperm nuclear vacuoles and sperm nuclear DNA damage (16, 17) or between sperm nuclear vacuoles and defects in chromatin remodeling during sperm maturation (6, 13, 15, 24). It is known that the integrity of sperm chromatin plays a key role in embryo development (37); in fact, faulty chromatin remodeling during spermiogenesis and oxidative stress may cause sperm DNA fragmentation and affect blastocyst development (38–40) and pregnancy outcomes (35, 41, 42). Thus, nuclear vacuoles can be correlated to the presence of sperm DNA fragmentation (16, 43), and this evidence further supports our hypothesis as the late, but not early, paternal effect has been shown to be related to sperm DNA fragmentation (35). Our retrospective, observational study confirms the extant data in the literature of a correlation between sperm vacuoles and negative IMSI outcomes. Our data also provide additional clinical information not previous available in the literature, such as for live-birth rates. Finally, the overall analysis of our data suggests that sperm vacuoles contribute to the late paternal effect. Thus, accurate analyses via IMSI of the presence and extent of sperm vacuoles should be considered an important aspect of achieving successful outcomes in ART cycles. However, further high-magnification studies to evaluate the influence the vacuolization pattern on clinical outcomes are required to demonstrate the importance of sperm impairments.

Acknowledgments: The authors thank Dr. Valentina Casciani, and Dr. Anna Maria Lobascio for their precious help in data analysis