Is the lack of prior exposure to sperm antigens associated with worse neonatal and maternal outcomes? A 10‐year single‐center experience comparing ICSI–TESE pregnancies to ICSI pregnancies

Nowadays, pathogenesis of preeclampsia (PE) is still unknown. Among the different etiological hypotheses, some authors proposed that it might be because of an abnormal immunologic response to a foreign fetal antigen derived from the father's spermatozoa. Indeed, the fetus is considered a semi‐allograft, being one half paternally derived in its antigenicity, and the first pathogenic insult of PE may be an abnormal maternal immune response toward this semi‐allogenic implant. In the context of artificial reproductive techniques, it has been shown that the use of donor and surgically retrieved spermatozoa (e.g., testicular sperm extraction [TESE]) increases the risk of PE, confirming the protective effect of sperm exposure on maternal complications.


INTRODUCTION
Even if most pregnancies have favorable outcomes, it is possible to face severe obstetric pathologies that lead to serious maternal and neonatal complications. 1 The great obstetrical syndromes include preeclampsia (PE), hypertensive disorders, intrauterine growth restriction (IUGR), gestational diabetes mellitus, and premature delivery. 2 Although in the last two conditions the pathophysiologic mechanism and possible interventions are clearer with respect to some decades ago, the real pathogenesis of PE and IUGR is still unknown, and therefore their only definitive cure is delivery, with the resolution of symptoms upon termination of pregnancy. 3 Despite vast research in the past years, the definition of PE is still based on a clinical diagnosis 4 : According to the American College of Obstetricians and Gynecologists, PE is diagnosed in the presence of new-onset hypertension accompanied by proteinuria. If proteinuria criteria are not met, the new-onset hypertension must be associated with signs of organ failure such as thrombocytopenia, renal or hepatic insufficiency, acute pulmonary edema, or new-onset headache. 5 Nowadays, PE is a major cause of morbidity and mortality in both mothers and neonates and complicates about 2%-8% of pregnancies worldwide. 6 Pregnant women affected by PE may develop potentially lethal complications, such as placental abruption, disseminated intravascular coagulation, hemorrhagic stroke, liver failure, acute kidney insufficiency, and, eventually, long-term cardiovascular morbidity. 7 Several risk factors are associated with an increased risk of developing PE, including the previous history of PE, maternal age > 40 years old, BMI > 35 kg/m 2 , nulliparity, multifetal gestations, pre-existing diabetes, pre-existing hypertension, chronic autoimmune diseases, and the presence of antiphospholipid autoantibodies. 8 The exact etiology of PE is not completely known. Some authors have proposed that PE has an immunological component. This is related to other risk factors such as limited sperm exposure 9 (in the case of barrier contraception and surgically retrieved spermatozoa), pregnancies achieved with donor insemination, and oocyte and embryo donation. 10 The fetus is considered a semi-allograft, being one half paternally derived in its antigenicity. 11 The first pathogenic insult of PE may be an abnormal maternal immune response toward the semi-allogenic implant. 12.13 Several studies support the hypothesis that PE might be because of an abnormal immunologic response to a foreign fetal antigen derived from the father's spermatozoa. 14 With continuous and prolonged maternal exposure to the paternal antigens both during sexual intercourse and pregnancy, memory T cells eventually expand and induce a paternal antigen-specific tolerance. 15 In the context of artificial reproductive techniques (ART), the use of spermatozoa from a donor increases the risk of PE, because of the negative exposure history to those paternal antigens. 16 As for sperm donors, also the use of spermatozoa surgically retrieved from the testis (e.g., testicular sperm extraction-TESE) increases the risk of PE, 10 confirming moreover the protective effect of sperm exposure on maternal complications.
Consistently with the previously stated hypotheses, the aim of our study is to determine if the lack of exposure to sperm antigens is associated with worse maternal and neonatal outcomes in pregnancies obtained after intracytoplasmic sperm injection (ICSI)-TESE for obstructive azoospermia (OA).

Study design and population
This was a single-center case-control retrospective study, focusing on all the first pregnancies obtained through ICSI-TESE for OA at Humanitas Fertility Center between January 1, 2010 and December 31, 2019.
Those pregnancies were achieved with both fresh and frozen embryo transfer.
The cases were matched with controls in a 1:2 ratio and the inclusion and exclusion criteria were as follows: • The case group included women that did not have any previous exposure to the spermatozoa of their current partner, because of the male obstructive etiology. Therefore, all couples that had a previous pregnancy or miscarriage, either spontaneous or through ART in our or a secondary center, were excluded. • In addition, patients with either pre-gestational hypertension or diabetes mellitus type 1 were ruled out in order to limit potential confounders. No age limitations were taken into consideration. • The control group included patients that achieved their first pregnancy through cycles of ICSI using ejaculated spermatozoa, for a diagnosis of infertility other than azoospermia, in the same time frame at Humanitas Fertility Center. ICSI was performed either on fresh or cryopreserved oocytes and was followed by fresh embryo transfer.
The matching was performed taking into consideration: 3. year of controlled ovarian stimulation (COS).

Data collection and follow-up
The information collected for the database was retrieved from the Fertility Center internal web-based registry, in which patients' data are safeguarded by advanced threat prevention, enterpriseclass encryption, and any user needs to periodically renew their password.
Routinely, pregnant women were followed up during the gestation This procedure was carried out before initiating the COS on the female partner, in order to avoid the risk of not finding any spermatozoa at the time of oocytes retrieval, and the testicular spermatozoa was cryopreserved following a rapid two-phase protocol until the time thawing and using for ICSI. 23

Controlled ovarian stimulation
According to the anti-Müllerian hormone (AMH) levels and antral follicular count, patients were stratified into different categories for the prediction of ovarian response to the ovarian stimulation and, in accordance with the Poseidon criteria, 24 a specific protocol was initiated: GnRH antagonist protocol, GnRH agonist long protocol, GnRH agonist short protocol, and flare-up GnRH agonist protocol. 25 During

Intracytoplasmic sperm injection
Both fresh and cryopreserved oocytes were used for ICSI. Once aspirated from the ovarian follicles, fresh oocyte-cumulus complexes were then treated by the biologist in several media, in order to prepare the oocyte for the injection. Cumulus cells and corona radiata were removed by transferring oocytes into an M2 medium with hyaluronidase 1 mg/mL for 1 min.
On the other hand, cryopreserved oocytes were properly warmed and treated, and then transferred into an injection dish for the procedure.
A morphologically normal and motile spermatozoon was isolated, either from cryopreserved testicular spermatozoa for the case group or from fresh semen for the control group, immobilized and then aspirated from the tail into the tip of the microinjection pipette. The oocyte was held by the holding pipette, and the spermatozoon was injected into the oocyte cytoplasm 26 with the microinjection needle.
The injection pipette was withdrawn gently and the oocyte was released from the holding pipette. The decision on how many embryos to transfer was taken according to the 2017 American Society of Reproductive Medicine guidelines. 28 The procedure was performed in the operating room in sterile conditions without the need of any anesthesia or analgesia. Accordingly, one or two embryos were released using a set of catheters of 1-1.5 cm from the uterine fundus under pelvic ultrasound visualization. 29 In frozen embryo transfers, endometrial synchronization was established before the transfer procedure through three different protocols: natural cycles, modified natural cycles with urinary hCG triggering, and artificial replacement cycles, as already described by De Cesare et al. 30

Outcomes' definitions
The primary outcome measure was the delivery rate, which was defined as the number of deliveries divided by the total number of clinical pregnancies.
The secondary outcomes focused on obstetric and neonatal complications.
The obstetric outcomes were the miscarriages (both first and second trimesters) rate, the frequencies of live birth and stillbirth, and the rate of the main obstetric complications such as pre-eclampsia, gestational hypertension, gestational diabetes, placenta previa, placental abruption, and premature rupture of membranes (PROM). The miscarriage rate was described as the number of clinical pregnancies who failed to continue beyond 12 weeks of gestation divided by the total number of clinical pregnancies. A live birth was defined as the delivery of a living baby after at least 22 weeks of gestation, whereas stillbirth was described as the death of a fetus prior to delivery after 28 weeks of gestation. 31 The neonatal outcomes are gestational age, prematurity rate, birth weight and its subcategories (appropriate for gestational age [AGA], small for gestational age [SGA], and large for gestational age [LGA]), sex ratio, 1-and 5-min APGAR, and the rate of congenital defects. A birth was defined at term if delivery occurred between 37 and 42 weeks of gestation; a preterm birth is defined as a delivery occurring before 37 weeks of gestation and, more specifically, it is defined as moderate-to-late preterm when it occurs between 32 and 37 weeks; very preterm between 28 and 32 weeks; and extremely preterm less than 28 weeks of gestation. According to the birth weight, growth percentiles were calculated using Medicine Fetal Barcelona Calculator and newborns were divided into three categories: AGA (between 10th and 90th percentile), SGA (<10th percentile), and LGA (>90th percentile). Macrosomia was defined as birth weight exceeding 4000 g. The APGAR score was included in order to objectively report the overall status of the neonates immediately after birth and their response to resuscitation, if performed. An APGAR score between 7 and 10 indicates an excellent status, with a score of 4-6 moderately depressed and a score of 0-3 severely depressed.

Ethical approval
Patients had previously signed a written informed consent regarding the use of their medical records for research purposes, as long as their anonymity was preserved and confidentiality of the medical record was guaranteed.
The study was approved on March 23, 2021 by the Independent Ethical Committee of the Humanitas Institutional Clinic (Milan, Italy) (approval number 14/21), and it was registered on clinicalTrials.gov before full variables extraction and statistical analysis were conducted (registration number NCT04852237).

Statistical analysis
Data were described as number and percentage or as median and interquartile range, as appropriate.
Continuous variables were analyzed with the Wilcoxon rank-sum test, whereas dichotomous variables were with a chi-squared test. All variables were analyzed by univariable logistic regression, and variables with a p value lower than 0.2 were submitted to multivariable regression analysis, in order to identify the possible factors associated with the outcomes. Logistic regression results were reported as an odds ratio (OR) and 95% confidence interval.
A p value lower than 0.05 was considered significant.

Population description
A total of 113 pregnancies occurring between January 1, 2010 and December 31, 2019, at Humanitas Fertility Center fulfilled the inclusion and exclusion criteria and were considered cases. These were matched with a ratio of almost 1:2 to a control group, based on female age at cycle induction, BMI classes, and year of COS; a total of 214 patients were included in controls.
As it is possible to see from On the other hand, the etiologies of acquired causes of OA most likely arose before initiating any sexual relationship with their current partners.
The most common acquired causes were idiopathic (proximal or distal obstruction without specified etiology) (54.6%), iatrogenic (related to urological or abdominal surgeries, mainly bilateral hernia repair, vasectomy, and varicocoelectomy) (31.8%), and post-infective (related to male accessory glans inflammation) (13.6%).  Table 3 depicts the pregnancy outcomes in the two populations.

Pregnancy outcomes
The first trimester miscarriage rate was definitely higher in the cases (13.27%) than in controls (6.07%), and this difference was statistically significant (p = 0.027). There was no difference between the rate of ectopic pregnancies (1.77% vs. 0.93%, p = 0.611) of the two groups. Univariate logistic regression analysis was additionally performed to determine the relationship between the investigated variables and miscarriage as outcome, as seen in

Maternal complications
The maternal complications can be appreciated in Table 6. Overall, 22 complications were recorded in cases and 45 complications in controls; no statistically significant difference was observed (p = 0.989).
Univariate logistic regression analysis was performed to determine the relationship between the investigated variables and maternal complications, but no significant impact was underlined, as reported in Table 7.

Neonatal complications
The neonatal population was differentiated into singleton pregnancies and multiple pregnancies: Overall, the singleton gestations were 77 vs.
In addition, newborns belonging to both groups had an overall excellent status at delivery, as indicated by median 1 and 5-min APGAR scores.
Table9 shows the neonatal population of twin pregnancies.
In total, 38 newborns were alive at birth in cases, whereas one stillbirth occurred at 32 weeks of gestation; in controls, all twins were alive at birth, but one neonate shortly died after delivery because of Potter's syndrome, a rare fatal condition related to anidramnios. 1 min 9 (9-9) 9 (9-9) 0.632 5 min 10 (9-10) 10 (10-10) 0.136 Note: Results shown as number and percentage. Abbreviations: AGA, appropriate for gestational age; LGA, large for gestational age; SGA, small for gestational age.
There was no significant difference in the rate of congenital malformations (7.96% vs. 4.68%, p = 0.226) between the 113 newborns of cases and 235 of controls, as shown in Table 10.
Newborns of the case group had congenital defects regarding the following districts: face (asymmetry of the mandible and palate); ears, nose, and throat (congenital bilateral deafness and pharyngeal immaturity); thorax (thymic hyperplasia); urinary tract (bilateral pyelectasis); genital tract (testicular atrophy and congenital anomalies of the vas deferens); musculoskeletal (clubfoot); and skin (angioma on the tight).
Newborns of the control group presented congenital malformations affecting the following: skull (turricephaly and plagiocephaly); ears (preauricular appendix); heart (unspecified anomalies of the valves); gastrointestinal tract (anorectal malformations); urinary tract (bilaterally enlarged kidneys = bilateral pyelectasis and ureteral anomalies); genital tract (congenital urachal anomaly and unilateral cryptorchidism); musculoskeletal (clubfoot and congenital hip dysplasia); and skin (angioma). In addition, a neonate had Potter's syndrome and shortly died after birth. One had an umbilical cord with a single umbilical artery.

DISCUSSION
The objective of this retrospective case-control study was to determine whether the lack of exposure to sperm antigens was associated with worse maternal and neonatal outcomes in pregnancies obtained after ICSI-TESE for OA.
The results showed that, by comparing two different populations of patients with similar basal characteristics, there was a higher miscarriage rate in cases with respect to controls. Once the pregnancy was established and evolved beyond the 12 weeks of gestation, the delivery rate was higher in controls with respect to cases. Regarding the maternal and neonatal complications, no substantial differences were found.
To the best of our knowledge, this is the first study underlying a higher risk of miscarriage in patients diagnosed with OA. Other studies mainly focused on the comparison between the type of azoospermia (OA vs. NOA): According to Pasqualotto et al., 34,35 NOA patients have an increased risk of abortion in respect to OA patients; instead, Bocca et al. 36 and Celikten et al. 37 did not find any significant difference in miscarriage rate between the two groups.
Ghazzawi et al. 38 found a significant increase in abortion rate in the testicular NOA group in respect to ejaculate and epididymal OA groups.
Taking into consideration the site of sperm extraction in OA (testicular vs. epididymal), Kamal et al. 39 did not find any significant difference between the two; also, when analyzing the specific etiology of obstruction, congenital vs. acquired, there was no difference in the site of sperm aspiration/biopsy. Instead, other studies 34,40 found that miscarriage rates were higher in cases that used spermatozoa retrieved by TESE, in comparison with retrievals by percutaneous epididymal sperm aspiration.
After investigating the different causes of OA (CBAVD, vasectomy, non-infective, infective/inflammatory and ejaculatory), Nicopollous et al. found a negligible disparity in early abortion rates across the five groups. 41 The ectopic pregnancy rate was comparable among our groups, inline with one study found in the literature. 42 Despite the ambiguity surrounding the pathogenesis of PE, one hypothesis proposes the maternal immune intolerance toward the foreign fetal antigen derived from the father's spermatozoa. Therefore, among infertile couples, patients whose male partners are diagnosed with azoospermia are thought to be at a greater risk of gestational hypertension and PE.
Nulliparous women are at a higher risk of developing PE in respect to multiparous women, but, in the case of new partner, the risk of the latter group returns similar to the risk during a first pregnancy. 43 The protective effect of pregnancy is seen also with both spontaneous and voluntary abortions. 44 In addition, multiparous women who become pregnant after at least 10 years from their previous pregnancy are as likely to have PE as nulliparous women. 45 Prolonged sexual cohabitation (≥ 12 months) and vaginal sperm exposure from the same partner reduce the risk of PE 46 ; moreover, the use of barrier contraceptive methods, such as condoms, increases the risk of PE. 47 In addition, it has been shown that pre-conceptual oral exposure to semen decreases the incidence of PE, thanks to the induction of oral tolerance. 48 The results of the present study showed that cases did not have a higher risk of developing gestational hypertension and PE with respect to controls. In addition, also the rate of other obstetrical complications (gestational diabetes, placenta previa, placental abruption, and PROM) was analyzed and no difference between the two groups was found.
When maternal complications were studied through univariate regression analysis, none of the considered variables seemed to have a statistically significant impact.
To the best of our knowledge, only Wang et al. 10  Probably the sexual intercourse in our couple during a spontaneous search of pregnancy with the same partner allowed sensitization to the partner's sperm antigens as well as in couples who performed ICSI from ejaculated semen. This hypothesis could be on the basis of the absence of increased risk of preclampsia in couples who perform ICSI by TESE.
The contrast of findings between this study and the aforementioned one could be explained by a selection bias/collider bias. Indeed, as the cases had a higher miscarriage rate and a lower delivery rate with respect to controls, the cases who experienced early termination of their pregnancy did not have the possibility to develop PE and other adverse outcomes. This was on the basis of the "smoking preeclampsia paradox", explained by Luque-Fernandez et al. 49 : After that several studies reported a reduced association between cigarette smoking and PE, this study showed how, by considering gestational age as a collider, the association between PE and smoking was biased. Indeed, smoking was associated with miscarriage, fetal death, and preterm delivery and, therefore, pregnant women who actively smoked and experienced early pregnancy termination or premature birth did not actually had the possibility to develop PE.
In relation to the neonatal outcomes, our study showed that singletons and twins born after ICSI with testicular spermatozoa from patients with OA had similar perinatal outcomes in comparison to the ones born with ICSI and ejaculated spermatozoa. As it is well-recognized that singletons and twins have different perinatal outcomes, separate analyses were carried out between these two subgroups. Overall, no differences were found among basal characteristics and complication rates between the two groups, with the exception of a higher prematurity rate in twins of the control group.
Our finding was in-line with the current literature findings; indeed, studies showed that neonatal outcomes of children conceived with non-ejaculated spermatozoa are equivalent to those of newborns conceived with ejaculated spermatozoa. Belva et al. 50 compared pregnancies obtained from non-ejaculated spermatozoa (both testicular and epididymal, for NOA and OA, respectively) with those obtained with ejaculated spermatozoa, and the only significant finding was those twins from non-ejaculated spermatozoa had a greater risk of perinatal death, but not of stillbirth.
In our case-control study, the male-to-female ratio was similar, and also the rate of congenital defects did not vary between neonates and found that the overall rate of congenital malformation was comparable among populations, but the rate of congenital heart defects was greater in singleton boys conceived with non-ejaculated sperm, and the risk of undescended testicles increased in both singleton and twin boys according to the severity of male infertility. 52 The primary strength of this study is that, to the best of our knowledge, it is one of the first studies focusing on miscarriage and maternal complications rates in couples diagnosed with OA. In addition, the randomization of age and BMI controls allowed to reduce confounding factors. Furthermore, as the study was limited to a single center, all the interventions were performed by the same andrologists, reproductive gynecologists, and embryologists. At the same time, if a multicenter investigation was conducted, a larger sample size could have been studied.
Regarding possible study limitations, worth mentioning is the retrospective design of the study: Some data could have erroneously been recorded or reported by the patients, although the data retrieved were from the discharge letters obtained from the patients and our center's internal web-based database.

CONCLUSIONS
Generally, patients with surgically retrieved spermatozoa have lower chances to obtain a pregnancy. This study showed that, once couples diagnosed with OA achieve a pregnancy, they have a much higher risk of miscarriage in the first trimester in respect to non-azoospermic patients. Moreover, controls had a higher delivery rate in respect to cases; however, when the fetal status at birth was compared, no difference was found between live births and stillbirths.
Differently from the findings in the literature, no association with PE was found. This might be related to a collider bias/left truncation bias: As azoospermic patients are at higher risk of early termination of pregnancy, it results that they do not have the possibility to develop PE and other adverse outcomes.