Effect of Intracytoplasmic Sperm Injection Alone versus ICSI and Preimplantation Genetic Screening on Pregnancy Outcomes of Patients with A History of Gestational Trophoblastic Disease: A Retrospective Study

Abstract

Background:

Gestational trophoblastic disease (GTD) is characterized genetically by an excess paternal genome and maternal chromosome loss. Intracytoplasmic sperm injection (ICSI) and ICSI with preimplantation genetic screening (PGS) enhance the selection of viable embryos by ensuring that only those with the appropriate genetic makeup are implanted. We aimed to evaluate the pregnancy outcome following ICSI and ICSI/PGS in infertile women with a history of GTDs.

Materials and Methods:

In this retrospective study, we recruited couples who were referred to the Royan Institute with infertility complaints with GTD history from 2010 to 2022. GTD had been confirmed by serial β-human chorionic gonadotrophin (β-hCG) titer, ultrasonography, and histopathology assessment of the evacuated uterine contents. The fluorescent in situ hybridization (FISH) probes were specific for the chromosomes 13, 18, 21, X, and Y.

Results:

A total of 69 cycles of ICSI (n=41) and ICSI/PGS (n=28) were analyzed. The two treatment groups were comparable in terms of patients’ demographic characteristics. The mean number of total retrieved oocytes, MII oocytes, and obtained embryos in the ICSI/PGS cycle was higher compared to the ICSI cycle (P<0.001 for all). Also, a statistically significant difference between groups in the mean number of poor embryos (P=0.004) was found. No statistically significant difference was observed in clinical pregnancy, miscarriage, and live birth rates per embryo transfer (ET) between groups. The findings indicate that 27.2% of embryos exhibited genetic normalcy in the ICSI/PGS group.

Conclusion:

Despite achieving more embryos in ICSI/PGS cycles, the success rates of pregnancy in both groups are approximately the same. Both of these methods can be effective in the management of women with the previous GTD. Considering the high costs and the necessity to eliminate 46XX embryos during PGS procedures, it is advisable to restrict this method to cases with a significant risk of recurrent GTD.

Introduction

Gestational trophoblastic disease (GTD) is a heterogeneous neoplastic group of diseases characterized by abnormally proliferating trophoblastic tissues (1, 2). GTD manifests four main pathological features: hydatidiform mole (HM, complete or partial), invasive mole, choriocarcinoma, and placental site trophoblastic tumor (PTT) (3). Most of the GTDs are HM, encompassing the complete and partial HMs defined as excessive trophoblast proliferations with hydropic swelling of the chorionic villi (4, 5). Various risk factors have been identified as potential causes of GTD. Two of the most significant risk groups are women over 35-40 and female teenagers, affecting individuals across all regions and ethnic backgrounds (1, 2, 6). Additionally, factors such as a history of spontaneous miscarriage, a prior diagnosis of GTD, a family history of GTD, and blood type (specifically the ABO blood group) may be linked to the development of GTD (1, 7, 8). Notably, a previous spontaneous miscarriage can increase the risk of developing GTD by 2 to 3 times (1, 9).

Recurrent GTD is defined as having experienced at least two pregnancies affected by GTD (10). Women with a history of one GTD have an approximately 1-2% chance of recurrence in subsequent pregnancy; however, the recurrence rate is 20% higher after two GTD pregnancies (11, 12). Genetically complete hydatidiform mole (CHM) presents an excess paternal genome (dispermia: entrance of two sperms into one oocyte) and maternal chromosome loss, resulting in 46,XX embryo (90%) as the origin of androgenic chromosome, which mitochondrial DNA (mtDNA) has maternal derivation (7, 13). Moreover, the data obtained from the previous research indicates that the oocyte could potentially have a significant impact on the pathophysiology of recurrent hydatidiform mole, despite it being frequently overlooked (14). However, partial hydatidiform mole (PHM) arises from an ovum fertilized by two spermatozoa, leading to a triploid (a total of 69 chromosomes rather than the normal 46 chromosomes) conceptus (1).

The potential role of assisted reproductive technology (ART) in preventing recurrent GTD is noteworthy. Intracytoplasmic sperm injection (ICSI) proceeds through the assurance of fertilization by a single sperm (15, 16). Interestingly, however, following the ICSI method, several of the GTDs have been reported (17-22). Theoretically, ICSI with pre-implantation genetic screening (PGS) caused 46, XY embryos that could prevent recurrent GTD (12). In recent studies, biparental origin was proposed for GTD pathophysiology, which could be assessed by microsatellite polymorphisms on different chromosomes (7, 23, 24). Despite these insights, the efficacy of adding the PGS procedure to ICSI for the prevention of GTD remains unclear. In this study, we aimed to compare pregnancy outcomes of patients with GTD history who underwent ICSI and ICSI/PGS processes.

Materials and Methods

Subjects

In this retrospective study, we evaluated documents of all infertile women who were referred to the Royan Institute in Tehran, Iran, for infertility issues and had a history of one or more instances of GTD between the years 2010 and 2022. GTD had been confirmed by serial β-human chorionic gonadotrophin (β-hCG) titer, ultrasonography, and histopathology assessment of the evacuated uterine contents.

The evaluation of the couple’s karyotype and extensive genetic counseling are standard practices for the treatment of these patients at the Royan Institute. During consultations, patients were informed that to minimize the risk of recurrence of GTD, it is advisable to incorporate preimplantation genetic screening (PGS) alongside the ICSI protocol. The decision to proceed with either the ICSI cycle alone or in conjunction with PGS was ultimately determined by the patients with considering the cost of PGS.

In all patients, the ovarian stimulation was achieved by a standard long gonadotropin-releasing hormone agonist (GnRHa) down-regulation with recombinant follicle-stimulating hormone (rFSH). The standard procedure for ovarian stimulation and ovulation retrieval at Royan Institute has been thoroughly outlined in a previously published article (25). The ICSI procedure was routinely conducted on mature metaphase II oocytes to prevent dispermia, with the presence of two pronuclei (2PN) indicating successful fertilization. Information regarding the assessment of embryo quality, along with the procedures for freezing and thawing embryos at the Royan Institute, can be found in our earlier publication (26). All embryos progressed to the cleavage stage (day three post-retrieval), and in the PGS group, they underwent biopsy for single or two-cell PGS using the fluorescence in situ hybridization (FISH) technique.

FISH procedure

The FISH probes were specific for the chromosomes 13, 16 and 21 (Vysis, Inc, USA) to determine the ploidy, in addition to the probes allocated for chromosomes X and Y to provide a diploid 46, XY complement. The signals of FISH probes were evaluated under an upright fluorescence microscope (Olympus, DX50, Japan). Two distinct same color signals for each autosomal chromosomes and one for each sex chromosome, considered as normal. In addition to the exclusion of aneuploid embryos, it is preferable not to transfer female embryos, as well as those exhibiting micronuclei and extra-nuclei, due to the presence of multiple micronuclei.

Embryo transfer and pregnancy

In the ICSI group, up to three fresh or frozen good-quality embryos were transferred in the cleavage stage. In the PGS group, embryos were transferred on day five of identification, at least one compacted embryo or blastocyst with normal chromosomes. Pregnancy was confirmed by a persistent rise in serum β-hCG levels at day 14-16 after embryo transfer. Clinical pregnancy was considered by observing the gestational sac with a positive heartbeat at the 6-8 weeks after embryo transfer (ET) using a vaginal ultrasound scan. The medical records of the patients were examined, and a comparison was made between the two groups regarding their demographic characteristics, medical history, treatment records, and the outcomes of the treatment cycle.

Statistical analysis

Statistical analysis was performed using SPSS software (version 22; IBM Corp., Armonk, NY). Data were initially tested for normal distribution using the Kolmogorov-Smirnov test. Continuous variables were expressed as mean ± SD, and categorical variables as number (percentage). Quantitative variables with normal distribution were analyzed using the 2-tailed Student t tset for comparing in two groups, and the Chi-square test for comparing qualitative variables between two groups. A P<0.05 was considered statistically significant.

Ethical considerations

This retrospective study was approved by the Ethics Committee of Royan Institute (IR.ACECR.ROYAN. REC.1396.88).

Results

Demographic and clinical characteristics of the studied couples, categorized by the type of treatment cycle, are presented in Table 1. A total of 69 cycles of ICSI/PGS and ICSI were analyzed in this study, comprising 41 cycles (59.4%) of ICSI and 28 cycles (40.5%) of ICSI/PGS, conducted between 2010 and 2022. The mean age of women and their husbands, along with the body mass index and the average age of women in their first molar pregnancy, did not show a statistically significant difference between the two groups. The blood groups of women did not exhibit a significant difference between the two groups, with the majority of patients in both groups having blood group O (P=0.910). The hormonal profiles of women exhibited no statistically significant differences between the two groups. However, the mean level of TSH hormone was elevated in the ICSI/PGS group in comparison to the ICSI group. It is important to highlight that at Royan Institute, patients with abnormal TSH levels are routinely managed by an endocrinologist prior to the initiation of infertility treatment cycles. Significant differences were also found regarding the reason for infertility (P=0.002) and the history of previous pregnancy (P<0.001) between the ICSI and ICSI/PGS group. So that, the mean number of previous pregnancies was significantly different between the two groups (P<0.001). On the other hand, the frequency of patients with a history of 2 or more molar pregnancies in the ICSI/PGS group was significantly higher than in the ICSI group (P<0.001). The mean number of previous miscarriages, excluding molar pregnancies, was higher in the ICSI cycles compared to the ICSI/ PGS cycles, although this difference was not statistically significant (P=0.284).

Table 1.

Demographic and clinical characteristics of participants in two cycles of ICSI and ICSI/PGS

Variables		ICSI (n=41)	ICSI/PGS (n=28)	P value†
Age (Y)		33.68 ± 3.98	33.75 ± 4.89	0.950
Spouse age (Y)		37.85 ± 4.65	37.29 ± 5.85	0.664
Age at first GTD (Y)		27.16 ± 5.24	24.92 ± 3.32	0.067
BMI (kg/m2)		25.69 ± 3.95	25.89 ± 4.01	0.839
Blood groups				0.910
	A	8 (19.5)	4 (14.3)
	B	7 (17.1)	6 (21.4)
	O	24 (58.5)	17 (60.7)
	AB	2 (4.9)	1 (3.6)
Reasons of infertility				0.002*
	Female factor (ovulatory and tubal)	8 (19.5)	2 (7.1)
	Unexplained	6 (14.6)	7 (25.0)
	Male	9 (22)	1 (3.6)
	Recurrent abortion	5 (12.2)	14 (50)
	Mixed factor (Both male and female)	13 (31.7)	4 (14.3)
Regular menstrual		34 (82.9)	24 (85.7)	0.817
FSH (MIU/ML)		6.87 ± 3.47	6.37 ± 1.89	0.491
LH (MIU/ML)		4.39 ± 3.79	5.05 ± 5.07	0.571
AMH (ng/ml)		2.69 ± 2.78	1.85 ± 1.58	0.437
TSH (MIU/ML)		1.52 ± 0.93	2 ± 0.79	0.033*
PRL (MIU/ML)		150.93 ± 38.97	142.81 ± 49.06	0.643
Number of previous pregnancies		1.60 ± 0.94	2.64 ± 1.15	<0.001*
Number of previous miscarriages other than molar pregnancy		0.80 ± 0.46	0.64 ± 0.25	0.284
Family history of GTD		0 (0.0)	1 (3.8)	-
Number of previous histories of GTD				<0.001*
	Once	39 (95.1)	11 (39.3)
	≥ 2 times	2 (4.9)	17 (69.7)

Values are presented by mean ± SD and number (%). ICSI; Intracytoplasmic sperm injection, PGS; Preimplantation genetic screening, GTD; Gestational trophoblastic disease, BMI; Body mass index., FSH; Follicle-stimulating hormone, LH; Luteinizing hormone, AMH; Anti-Müllerian hormone, TSH; Thyroid stimulating hormone, PRL; Prolactin, †; P value is obtained by independent sample t test and Chi2 test, and *; P<0.05 was statistically significant.

Table 2.

Comparison of the consequences of ICSI and ICSI/PGS

Variables		ICSI (n=41)	ICSI/PGS (n=28)	P value†
Total No. of retrieved oocyte		6.60 ± 0.61	11.67 ± 1.23	<0.001*
No. of MII oocytes		5.26 ± 0.53	9.78 ± 0.87	<0.001*
No. of 2PN		3.04 ± 0.37	6.50 ± 0.70	<0.001*
Total number of obtained embryos		3.21 ± 0.37	5.39 ± 0.46	<0.001*
Quality of the obtained embryo
	Good	0.47 ± 0.13	0.43 ± 0.15	0.854
	Fair	2.22 ± 0.33	2.89 ± 0.48	0.242
	Poor	0.03 ± 0.03	0.59 ± 0.20	0.004*
No. of blastocyst-stage embryos		0.78 ± 0.32	0.93 ± 0.40	0.776
Quality of transferred embryos
	Good	0.41 ± 0.11	0.22 ± 0.11	0.244
	Fair	1.22 ± 0.18	1.26 ± 0.25	0.893
	Poor	0.0 ± 0.0	0.26 ± 0.11	0.016*
No. of transferred embryos		1.29 ± 0.71	1.03 ± 0.63	0.131
No ET cases		3 (7.5%)	5 (17.9%)	0.192
Clinical pregnancy/ET (%)		8/37 (21.6)	6/23 (26.08)	0.564
Missed abortion/ET (%)		2/37 (5.4)	3/23 (13.04)	0.298
Live birth /ET (%)		6/37 (16.2)	3/23 (13.04 )	0.577

Values are presented by mean ± SE and number (%). ICSI; Intracytoplasmic sperm injection, PGS; Preimplantation genetic screening, MII; Metaphase II, 2PN; Two pronuclear., No.; Number, ET; Embryo transfer, †; P value is obtained by independent sample t test and Chi2 test, and *; P<0.05 was statistically significant.

The comparison of outcomes between ICSI and ICSI/ PGS is illustrated in Table 2. The average number of oocytes retrieved during the ICSI/PGS cycle was significantly greater than that in the ICSI cycle (P<0.001). Additionally, the mean numbers of MII and 2PN in the ICSI/ PGS cycle surpassed those in the ICSI cycle (P<0.001 for all). There was a statistically significant difference in the mean number of embryos between ICSI/PGS and ICSI cycles (P<0.001).

Regarding the embryo quality, we found a statistically significant difference between the ICSI/PGS and ICSI cycles in the mean number of poor embryos (P=0.004). The mean number of ET was higher in the ICSI cycle than the ICSI/PGS cycle, with no evidence of statistically significant difference (P=0.131). No statistically significant difference was observed between the ICSI/PGS cycle and ICSI cycle, in terms of clinical pregnancy and live birth per ET rates (P=0.564 and P=0.577).

Table 3.

Results of chromosomal analyses of obtained embryos (n=103) in the ICSI/PGS cycles

Variables		n (%)
Chromosomal constitution at biopsy
	Normal	28 (27.20)
	Abnormal	75 (72.81)
Type of Chromosomal abnormality
	Trisomy	17(22.67)
	Monosomy	20(26.67)
	Triploid/Tetraploid	19(25.33)
	Combined abnormalities	14(18.66)
	No diagnosis	5(6.67)

ICSI; Intracytoplasmic sperm injection and PGS; Preimplantation genetic screening.

Table 3 shows the general results of embryo and chromosomal analyses in all 126 ICSI/PGS embryos. Most embryos were observed in the 8-cells stage (51.58%) and the 6-cells stage (26.2%). The findings indicate that a mere 27.2% of embryos exhibited genetic normalcy; the most common genetic problem diagnosed was monosomy.

Discussion

In the present study, there were no cases of molar pregnancy recurrence noted after treatment cycles of ICSI alone or in conjunction with PGS. Furthermore, the pregnancy and live birth rates were comparable between the two treatment groups. Some previous studies have outlined ART techniques aimed at preventing GTD (6, 12). While ICSI may reduce the risk of recurrent GTD, it is crucial to understand that in vitro fertilization (IVF) does not assure a successful normal pregnancy (9, 27). Consequently, Nickkho-Amiry et al. (28) determined that employing ICSI should safeguard against triploidy; nevertheless, retrospective evidence indicates that ART does not completely prevent molar pregnancies. Research indicates that molar pregnancies following ART may arise from the use of diploid sperm or digynic triploidy. Specifically, studies suggest that triploidy resulting from ICSI is often of digynic origin (29). Macas et al. (30) found that in cases involving severe sperm abnormalities, 33% of triploid zygotes were attributed to diploid sperm. However, there is a lack of comprehensive data on the risk factors contributing to triploidy formation (28). Recently, Nickkho-Amiry et al. (28) have reported that the incidence rate of molar pregnancy following fresh ICSI (1/4302) and fresh IVF (1/4333) was comparable. In agreement with their study, our earlier investigation at the Royan Institute revealed that there were 10 cases of GTD following 41,540 ICSI procedures, equating to a rate of 0.240 per 1000 cycles (31). Furthermore, they have highlighted the recurrence risk of molar pregnancy following a previous instance with ICSI, which varies significantly from 1 in 6 to 1 in 29. Notably, even at the lowest recurrence rate of 1 in 29, the risk remains substantially higher compared to spontaneous conceptions, which have a recurrence risk of 1 in 80 (28). The recurrence of hydatidiform pregnancy may occur by coincidence or be linked to clinical variables associated with ART, such as advanced maternal age and poor oocyte quality (28). The increased incidence of recurrence appears to be influenced by both ART practices and patient characteristics. Consequently, women with a history of confirmed triploid molar pregnancy should consider ICSI as a treatment option. Additionally, the potential risk of recurrence in patients who have experienced a mole through IVF or ICSI suggests that PGD should be contemplated in subsequent cycles to mitigate the risk of recurrence (28). PGS can prevent transferring paternal isodisomy, the main format (CHM), by selecting only male embryos and verifying the ploidy status of the embryos to avert the occurrence of partial moles (12). Although nearly two decades have passed since the application of ICSI/PGS for GTD prevention (12), there is a lack of convincing evidence in favor of or against the use of ICSI/PGS for patients with a GTD history. According to Zhou et al. (32), a complete molar pregnancy that arises from the transfer of a euploid embryo underscores the necessity of comprehending the limitations of genetic testing platforms when dealing with embryos originating from abnormally fertilized oocytes.

In the present study, although the number of oocytes, MII, and embryos acquired during the ICSI/PGS protocol was notably higher than that achieved with the ICSI-alone method, probably due to increased oocyte stimulation as determined by the physicians; however, the embryo transfer rate did not show a significant increase in the ICSI/ PGS cohort. Although ICSI/PGS can prevent aneuploidy embryos from being transferred and could be expected to cause a more successful implantation rate and pregnancy outcome (33), both groups lead to similar conception rates and embryo quality, indicating that ICSI/PGS does not appear to improve pregnancy or GTD rate. In the total ICSI/PGS process evaluation, from the 126 obtained embryos, the FISH process was performed for 103 embryos, leading to 28 (27.20%) normo-chromosomal embryos. The highest detected abnormality in the embryos was monosomy. On the other hand, an analysis of the costs associated with the two aforementioned procedures revealed that the ICSI/PGS procedure was approximately 3.5 times more expensive than ICSI for women with a prior history of one GTD, whereas live births were more predominant in the ICSI alone procedure. Since in our study, no cases of molar pregnancy recurrence were observed in both groups, we recommend that ICSI is preferred over the ICSI/PGS procedure for preventing GTD. In addition, fewer chromosomal abnormality is expected in ICSI/PGS as this method eliminates aneuploid embryos. Nevertheless, considering the heavy cost of this method, compared to ICSI, one might ignore the merits of ICSI with PGS.

In this study, familial abortion and GTD history were observed only in one woman who had a history of three GTDs. Recurrent GTD is infrequent (27), and the familial cases are particularly uncommon (8). Linkage analysis has demonstrated that in most families, two maternaleffect genes including NLRP7 (NOD-like receptor pyrindomain-containing 7 is involved in the regulation of immune responses) and KHDC3L (KH domain containing 3-like is related to the oocyte cytoskeleton.) NLRP7 and KHDC3L are critical for normal trophoblast development, and mutations NLRP7 and KHDC3L, resulting in abnormal oocytes and developing hydatidiform moles gestational abnormalities (34, 35). Changes in the expression of maternal mitochondrial genes have been linked to GTD, suggesting that the oocyte may significantly influence the pathophysiology of recurrent hydatidiform mole (23). Consequently, utilizing donor oocytes in IVF/ICSI could serve as a potential treatment to avert recurrent GTD and facilitate a successful pregnancy (14).

It should be noted that one of the limitations of the study is that PGS was done by the FISH method, so these results cannot be generalized to other methods of PGS, and further studies with a larger sample size are recommended. It is important to highlight that the two groups differed in terms of recurrent GTD history. This patient selection bias, resulting from the doctor’s choice of treatment, was inevitable and represents a limitation of the retrospective study. Hence, randomized clinical trials are essential in this context to compare the efficacy of these treatment approaches.

Conclusion

Despite achieving more embryos in ICSI/PGS cycles, the success rate of pregnancy in both ICSI and ICSI/PGS groups is approximately the same. Both of these methods can be effective in the management of women with the previous GTD. Chromosomal euploidy and the prevention of uniparental disomy in ICSI/PGS are anticipated outcomes; however, the expenses associated with this method can be significantly burdensome. Considering the high costs and the necessity to eliminate 46XX embryos during PGS procedures, it is advisable to restrict this method to cases with a significant risk of recurrent GTD.

Conflict of Interest

Maryam Hafezi, Samira Vesali, Hamid Gourabi are reviewers for this journal. They did not participate in any capacity related to the peer review of this manuscript, nor were they involved in editorial decisions.

Author’s Contributions

Z.Ch., F.Sh.A., N.Z.; Data curation. Z.Ch.; Writing-original draft, Investigation, and Visualization. M.H.; Conceptualization, Project administration, and Supervision. S.V.; Formal analysis. Z.Ch., M.H., N.Z., S.V.; Writing-reviewing and Editing. H.G., Supervision and Investigation. All authors read and approved the final version of the manuscript.