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Comparison of fresh and frozen ejaculated spermatozoa in sibling oocyte recipient cycles

Published:October 03, 2021DOI:https://doi.org/10.1016/j.rbmo.2021.09.020

      Abstract

      Research question

      Do IVF and intracytoplasmic sperm injection cycles using fresh and frozen ejaculated spermatozoa result in similar pregnancy outcomes in couples with non-male factor infertility?

      Design

      Retrospective cohort study; patients undergoing donor egg recipient cycles, in which oocytes from a single ovarian stimulation were split between two recipients, were reviewed. Two recipients of oocytes from a single donor were paired and categorized based on the type of ejaculated spermatozoa (fresh/frozen). Outcomes included delivery rate, implantation, pregnancy, pregnancy loss and fertilization rates.

      Results

      Of the 408 patients who received oocytes from a split donor oocyte cycle, 45 pairs of patients used discrepant types of ejaculated spermatozoa and were included in the study. Fertilization rate: fresh (74.8%); frozen (68.6%) (P = 0.13). Pregnancy rate: fresh (76%); frozen (67%); delivery rate: fresh (69%); frozen (44%); implantation rate was significantly higher: fresh (64%); frozen (36%) (P = 0.04). Rate of pregnancy loss was significantly higher in the frozen group compared with the fresh group (33% versus 5.9%, P = 0.013). Adjusted odds for delivery was 67% lower in the frozen group (95% CI 0.12, 0.89). Adjusted odds of pregnancy (adjusted OR 0.67, 95% CI 0.20, 2.27) and implantation (adjusted OR 0.5, 95% CI 0.12, 2.12) were not significantly different between the frozen and fresh sperm groups.

      Conclusion

      : In this model that controls for oocyte quality by using paired recipients from the same donor, frozen ejaculated spermatozoa resulted in lower delivery rates than those using fresh spermatozoa

      KEYWORDS

      Introduction

      Intracytoplasmic sperm injection (ICSI) gives couples with male factor infertility the chance of a reproductive future (
      • Aboulghar M.A.
      • Mansour R.T.
      • Serour G.I.
      • Fahmy I.
      • Kamal A.
      • Tawab N.A.
      • Amin Y.M.
      Fertilization and Pregnancy Rates after Intracytoplasmic Sperm Injection Using Ejaculate Semen and Surgically Retrieved Sperm.
      ;
      • Palermo G.D.
      • Schlegel P.N.
      • Hariprashad J.J.
      • Ergün B.
      • Mielnik A.
      • Zaninovic N.
      • Veeck L.L.
      • Rosenwaks Z.
      Fertilization and Pregnancy Outcome with Intracytoplasmic Sperm Injection for Azoospermic Men.
      ). Although ICSI was initially developed to treat severe male infertility, its association with improved fertilization rates regardless of semen characteristics has led to its widespread use (
      • Farhi Jacob
      • Cohen Kfir
      • Mizrachi Yossi
      • Weissman Ariel
      • Raziel Arieh
      • Orvieto Raoul
      Should ICSI Be Implemented during IVF to All Advanced-Age Patients with Non-Male Factor Subfertility?.
      ).
      Cryopreserved spermatozoa are frequently used in IVF cycles owing to its many benefits. For example, frozen spermatozoa avoids the need for multiple surgical procedures in patients with male factor infertility (
      • Kalsi Jas
      • Yau Thum M.
      • Muneer Asif
      • Pryor John
      • Abdullah Hossam
      • Minhas Suks
      Analysis of the Outcome of Intracytoplasmic Sperm Injection Using Fresh or Frozen Sperm.
      ) and allows for more flexibility in treatment planning (
      • Oates R.D.
      • Lobel S.M.
      • Harris D.H.
      • Pang S.
      • Burgess C.M.
      • Carson R.S.
      Efficacy of Intracytoplasmic Sperm Injection Using Intentionally Cryopreserved Epididymal Spermatozoa.
      ). Cryopreservation and subsequent thawing of spermatozoa has, however, been associated with poorer sperm morphology (Verheyen et al., 1997), sperm nuclear DNA damage (
      • Thompson-Cree M.E.M.
      • McClure Neil
      • Donnelly Eilish T.
      • Steele Kristine E.
      • Lewis Sheena E.M.
      Effects of Cryopreservation on Testicular Sperm Nuclear DNA Fragmentation and Its Relationship with Assisted Conception Outcome Following ICSI with Testicular Spermatozoa.
      ) and formation of free radicals (
      • Chatterjee S.
      • Gagnon C.
      Production of Reactive Oxygen Species by Spermatozoa Undergoing Cooling, Freezing, and Thawing.
      ). This raises the question of whether using cryopreserved spermatozoa results in poorer IVF outcomes compared with fresh sperm specimens.
      Previously published studies have shown that fresh and frozen spermatozoa lead to comparable outcomes in IVF-ICSI cycles; however, many of these studies were carried out in patients with male factor infertility or with the use of surgically retrieved spermatozoa (
      • Thompson-Cree M.E.M.
      • McClure Neil
      • Donnelly Eilish T.
      • Steele Kristine E.
      • Lewis Sheena E.M.
      Effects of Cryopreservation on Testicular Sperm Nuclear DNA Fragmentation and Its Relationship with Assisted Conception Outcome Following ICSI with Testicular Spermatozoa.
      ;
      • Ulug Ulun
      • Bener Faruk
      • Karagenc Levent
      • Ciray Nadir
      • Bahceci Mustafa
      Outcomes in Couples Undergoing ICSI: Comparison between Fresh and Frozen–Thawed Surgically Retrieved Spermatozoa.
      ;
      • Kalsi Jas
      • Yau Thum M.
      • Muneer Asif
      • Pryor John
      • Abdullah Hossam
      • Minhas Suks
      Analysis of the Outcome of Intracytoplasmic Sperm Injection Using Fresh or Frozen Sperm.
      ;
      • Yu Zhe
      • Wei Zhewen
      • Yang Jun
      • Wang Tao
      • Jiang Hongyang
      • Li Hao
      • Tang Zhe
      • Wang Shaogang
      • Liu Jihong
      Comparison of Intracytoplasmic Sperm Injection Outcome with Fresh versus Frozen-Thawed Testicular Sperm in Men with Nonobstructive Azoospermia: A Systematic Review and Meta-Analysis.
      ). Data comparing fresh and frozen spermatozoa in patients with non-male factor infertility using ejaculated spermatozoa, however, are limited. Many of the studies that specifically evaluated IVF-ICSI outcomes using ejaculated spermatozoa were also carried out in patients with male factor infertility (
      • Van Steirteghem A.
      • Nagy P.
      • Joris H.
      • Janssenswillen C.
      • Staessen C.
      • Verheyen G.
      • Camus M.
      • Tournaye H.
      • Devroey P.
      Results of Intracytoplasmic Sperm Injection with Ejaculated, Fresh and Frozen-Thawed Epididymal and Testicular Spermatozoa.
      ;
      • Kuczyński W.
      • Dhont M.
      • Grygoruk C.
      • Grochowski D.
      • Wołczyński S.
      • Szamatowicz M.
      The Outcome of Intracytoplasmic Injection of Fresh and Cryopreserved Ejaculated Spermatozoa—a Prospective Randomized Study.
      ;
      • Schachter-Safrai Natali
      • Karavani Gilad
      • Levitas Eliahu
      • Friger Michael
      • Zeadna Atif
      • Lunenfeld Eitan
      • Har-Vardi Iris
      Does Cryopreservation of Sperm Affect Fertilization in Nonobstructive Azoospermia or Cryptozoospermia?.
      ). In a retrospective study that compared fresh and frozen ejaculated spermatozoa in IVF-ICSI cycles using normozoospermia,
      • Borges Edson
      • Rossi Lia Mara
      • Victor Locambo de Freitas Christiany
      • Guilherme Patrícia
      • Carvalho Tatiana
      • Bonetti S.
      • Iaconelli Assumpto
      • Pasqualotto Fabio Firmbach
      Fertilization and Pregnancy Outcome after Intracytoplasmic Injection with Fresh or Cryopreserved Ejaculated Spermatozoa.
      found no difference in pregnancy outcomes. This study, however, did not adequately control for oocyte quality and was limited by a small sample size.
      Assisted reproductive technology (ART) using donor sibling-oocyte recipients (oocytes from the same donor stimulation transferred to two different recipients) offers a unique model that controls for oocyte quality and the inherent heterogeneity of stimulation cycles to the greatest degree possible. The aim of the present study was to determine if fresh and frozen ejaculated spermatozoa are associated with similar pregnancy outcomes in paired donor egg recipients that received oocytes from the same donor from the same stimulation in IVF-ICSI cycles in couples with non-male factor infertility.

      Materials and methods

       Inclusion and exclusion criteria

      The Institutional Review Board at Weill Cornell Medical College approved the present study protocol on 5 June 2020 (reference number: 19-06020283). For this retrospective cohort study, all patients initiating IVF cycles with anonymous donor oocytes at the Ronald O Perelman and Claudia Cohen Center for Reproductive Medicine between January 2010 and December 2016 were assessed for potential inclusion. Fresh embryo transfers were carried out in all cycles. Only cycles in which oocytes from a single ovarian stimulation cycle of a donor were split between two recipients were included. The two recipients of the single donor oocyte cycle were paired and categorized based on type of ejaculated spermatozoa. The two categories evaluated were fresh ejaculated spermatozoa and frozen ejaculated spermatozoa. Frozen spermatozoa were either from the patient's partner or an anonymous donor, which were provided by sperm banks. All spermatozoa used for ICSI had normal semen analyses. Spermatozoa that had semen analyses with suboptimal spermatozoa were not included. Patients with uterine and male factor infertility, including the use of surgically retrieved spermatozoa, were excluded.

       Clinical and laboratory protocols

      Ovarian stimulation, trigger for final oocyte maturation, oocyte retrieval, embryo culture and embryo transfer were carried out in accordance with the clinic's standard protocols (
      • Huang Jack Yu Jen
      • Rosenwaks Zev
      Assisted Reproductive Techniques.
      ). Anonymous oocyte donors were started on oral contraceptive pills (Ortho-Novum) (Janssen Pharmaceuticals, Beerse, Belgium) for pretreatment follicular synchronization. Ovarian stimulation was carried out to maximize follicular response while minimizing the risk of ovarian hyperstimulation syndrome. The initial gonadotrophin dose was based on age, weight, antral follicle count, AMH and previous response to stimulation. Ovarian stimulation was carried out with a combination of gonadotrophins: Follistim (Merck, Kenilworth, NJ, USA); Gonal-F, (EMD-Serono, Geneva, Switzerland); Menopur (Ferring Pharmaceuticals Inc, Parsippany, NJ, USA), or all three, with ovulation being suppressed with once daily 0.25 mg Ganirelix Acetate (Merck, Kenilworth, NJ, USA) or Cetrotide (EMD-Serono, Geneva, Switzerland) injections (
      • Huang Jack Yu Jen
      • Rosenwaks Zev
      Assisted Reproductive Techniques.
      ).
      In most cycles, HCG was used as the ovulation trigger during the early years of the study period and gonadotrophin releasing hormone agonist triggers were incorporated into clinical practice in the latter years of the study period. Novarel (Ferring Pharmaceuticals Inc, Parsippany, NJ, USA) or Pregnyl (Merck, Kenilworth, NJ, USA) was administered according to a sliding scale (10,000 IU for oestradiol <1500 pg/ml, 5000 IU for oestradiol 1501–2500 pg/ml, 4000 IU for oestradiol 2501–3000 pg/ml and 3300 IU for oestradiol >3001 pg/ml) when given as the sole trigger (
      • Huang Jack Yu Jen
      • Rosenwaks Zev
      Assisted Reproductive Techniques.
      ). The trigger was given when the two lead follicles attained at least a mean diameter wider than 17 mm. Oocyte retrieval was carried out with transvaginal ultrasound guidance under conscious sedation about 35–36 h after trigger administration. All retrieved oocytes were exposed to 40 IU recombinant hyaluronidase (Cumulase) (Halozyme Therapeutics Inc., San Diego, CA, USA) to remove the cumulus–corona complex (
      • Palermo Gianpiero D.
      • Kocent Justin
      • Monahan Devin
      • Neri Queenie V.
      • Rosenwaks Zev
      Treatment of Male Infertility.
      ;
      • Palermo Gianpiero D.
      • Neri Queenie V.
      • Schlegel Peter N.
      • Rosenwaks Zev
      Intracytoplasmic Sperm Injection (ICSI) in Extreme Cases of Male Infertility.
      ). Test Yolk Buffer with Gentamicin (Irvine Scientific, California, USA) is used for sperm cryopreservation at our centre.
      In our centre, intracytoplasmic sperm injection is carried out in our centre for all patients undergoing IVF with donor oocytes. Sperm injection was carried out based on previously described protocols (
      • Palermo Gianpiero D.
      • Kocent Justin
      • Monahan Devin
      • Neri Queenie V.
      • Rosenwaks Zev
      Treatment of Male Infertility.
      ;
      • Palermo Gianpiero D.
      • Neri Queenie V.
      • Schlegel Peter N.
      • Rosenwaks Zev
      Intracytoplasmic Sperm Injection (ICSI) in Extreme Cases of Male Infertility.
      ). The spermatozoon selected for injection was based on head morphology, midpiece and flagellar shape and dynamic characteristics, such as swimming patterns and progression (
      • Palermo Gianpiero D.
      • Kocent Justin
      • Monahan Devin
      • Neri Queenie V.
      • Rosenwaks Zev
      Treatment of Male Infertility.
      ;
      • Palermo Gianpiero D.
      • Neri Queenie V.
      • Schlegel Peter N.
      • Rosenwaks Zev
      Intracytoplasmic Sperm Injection (ICSI) in Extreme Cases of Male Infertility.
      ). Oocytes were examined 12–17 h after ICSI for the presence of two distinct pronuclei and two clear polar bodies. All embryos were cultured using in-house culture media and were transferred on day-3 or day-5 (blastocyst). All embryo transfers were carried out with Wallace catheters (Smiths Medical Inc., Norwell, MA, USA) at approximately 1–2 cm less than the uterine depth identified at a previous trail transfer. Before embryo transfer, transvaginal ultrasound and hysterosalpingograms were carried out to evaluate the uterus. Saline infusion sonograms and hysteroscopies were also carried out if indicated.
      Recipients underwent programmed endometrial preparation based on previously described protocols (
      • Irani Mohamad
      • Reichman David
      • Robles Alex
      • Melnick Alexis
      • Davis Owen
      • Zaninovic Nikica
      • Xu Kangpu
      • Rosenwaks Zev
      Morphologic Grading of Euploid Blastocysts Influences Implantation and Ongoing Pregnancy Rates.
      ;
      • Melnick Alexis P.
      • Setton Robert
      • Stone Logan D.
      • Pereira Nigel
      • Xu Kangpu
      • Rosenwaks Zev
      • Spandorfer Steven D.
      Replacing Single Frozen-Thawed Euploid Embryos in a Natural Cycle in Ovulatory Women May Increase Live Birth Rates Compared to Medicated Cycles in Anovulatory Women.
      ), using leuprolide acetate in the luteal phase. Transdermal oestradiol patches (0.1 mg) were initiated on the second day of menses and serially increased up to a dose of 0.4 mg until an endometrial thickness of 7 mm or thicker was attained. Patients were started on progesterone on the night after donor trigger with a starting dose of 25 mg intramuscularly for 1 night and followed thereafter by 50 mg intramuscularly daily, and the oestradiol dose was decreased to 0.2 mg. Oestradiol and progesterone levels were measured after transfer to confirm the adequacy of the supplementation. Luteal support in recipients was continued until 10–12 weeks of gestation.

       Study variables

      Demographic and baseline characteristics recorded for all recipients include age at retrieval, body mass index (kg/m2), gravidity, parity, history of uterine surgery, peak endometrial thickness (mm), paternal age, number of embryos transferred and transfer day. All clinical pregnancies were confirmed using transvaginal ultrasonography, noting the number of gestational sacs and fetuses with cardiac activity. The primary outcome was delivery rate. Secondary outcomes included fertilization rate, implantation rate, positive pregnancy rate and rate of pregnancy loss. Fertilization rate was defined as the number of embryos with two pronuclei divided by the number of mature oocytes. Implantation rate was defined as the number of intrauterine gestational sacs seen via transvaginal ultrasonography divided by the number of embryos transferred for each patient. Positive pregnancy rate was defined as the number of positive beta HCG levels on CD28 (serum level >5 mIU/ml) per embryo transfer cycle. Any loss of a positive pregnancy before 24 weeks was considered a pregnancy loss. Specifically, biochemical pregnancy was defined as an early pregnancy loss after positive beta-HCG test without evidence of intrauterine pregnancy on ultrasound. An ectopic pregnancy was defined as a positive beta- HCG test with confirmed extrauterine pregnancy on ultrasound. Spontaneous miscarriage was defined as an early pregnancy loss after positive beta HCG test with confirmed intrauterine pregnancy on ultrasound. Any birth at 24 weeks of gestation or greater was considered in the delivery rate.

       Statistical analyses

      Descriptive statistics were used to characterize demographic and clinical factors of interest. Continuous variables are expressed as median (interquartile range) and categorical variables are expressed as n (%). Where appropriate, the chi-squared test (or Fisher's exact test) and the Wilcoxon rank-sum test were used to examine the association between demographic, clinical variables of interest and type of ejaculated spermatozoa (fresh or frozen). Type of spermatozoa and statistically significant variables identified by univariate analysis were included in mixed-effects logistic regression models to evaluate the independent effect on pregnancy, implantation and delivery rate. The number of pregnancy losses to run a multivariable model was insufficient. A mixed-effects logistic regression model was used to account for the clustered nature of the donor pairs. All P-values are two-sided with statistical significance evaluated at the 0.05 alpha level. R Version 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria) was used for all analyses.

      Results

      A total of 1013 patients who underwent donor egg recipient cycles were screened for inclusion, of which 408 patients had received oocytes from a split donor oocyte cycle. Of these 408 patients, 45 pairs of patients (90 recipients) used discrepant types of ejaculated spermatozoa (fresh spermatozoa or frozen spermatozoa). Baseline characteristics of the two groups are presented in Table 1. Of the 45 frozen sperm samples used, 30 (67%) were from an anonymous donor and the remaining 15 (33%) were from the recipient's partner. The median age of the recipients was 43 years in the fresh sperm group and 44 years old in the frozen sperm group (P = 0.62). The most common reason for infertility in each group was diminished ovarian reserve. The groups were similar for maternal age, paternal age, gravidity, parity, body mass index, history of prior uterine surgery, type of infertility, number of embryos transferred, peak endometrial stripe thickness and number of donor oocytes given to each recipient. The number of day-3 and day-5 transfers in the frozen sperm group (67% and 33%, respectively) compared with the fresh sperm group (60% and 40%, respectively) was not significantly different (P = 0.66). The fertilization rate was not statistically significant at 74.8% in the fresh sperm group and 68.6% in the frozen sperm group (P = 0.13).
      TABLE 1DESCRIPTIVE STATISTICS OF STUDY COHORT, STRATIFIED BY TYPE OF EJACULATED SPERMATOZOA
      Spermatozoa
      Fresh (n = 45)Frozen (n = 45)P-value
      Donor age, years28.0 (26.0–30.0)28.0 (26.0–30.0)>0.99
      Recipient age, years43.0 (42.0–46.0)44.0 (42.0–45.0)0.62
      Gravidity, n (%)0.62
      012 (27)14 (31)
      19 (20)14 (31)
      215 (33)8 (17.8)
      33 (6.7)2 (4.4)
      42 (4.4)3 (6.7)
      51 (2.2)2 (4.4)
      61 (2.2)2 (4.4)
      71 (2.2)0 (0)
      81 (2.2)0 (0)
      Gravid, n (%)33 (73)23 (51)0.05
      Parity, n (%)0.60
      033 (73)35 (78)
      110 (22)10 (22)
      22 (4.4)0 (0)
      Parous, n (%)14 (31)8 (18)0.22
      Uterine surgery, n (%)30 (67)21 (47)0.09
      Body mass index23.2 (21.3–26.1)22.8 (21.3–25.3)0.74
      Infertility diagnosis, n (%)0.63
      Diminished ovarian reserve41 (91.1)43 (95.6)
      Endometriosis1 (2.2)1 (2.2)
      Tubal2 (4.4)0 (0)
      Anovulatory0 (0)1 (2.2)
      Idiopathic1 (2.2)0 (0)
      Number of previous IVF attempts,

      n (%)
      0.01
      Statistically significant.
      09 (20)8 (17.8)
      1–215 (33.3)8 (17.8)
      3–517 (37.8)17 (37.8)
      6–104 (8.9)9 (20)
      >100 (0)3 (6.7)
      Sperm source<0.001
      Statistically significant.
      Anonymous donor0 (0)30 (67)
      Partner fresh jaculate45 (100)0 (0)
      Partner frozen0 (0)15 (33)
      Partner age43 (39, 48)45 (41, 48)0.45
      Unknown811
      Endometrial thickness, mm10.20 (9.50–11.20)10.40 (9.00–11.80)0.85
      Transfer day, n (%)0.66
      327 (60)30 (67)
      518 (40)15 (33)
      Embryos transferred, n (%)0.13
      113 (29)7 (16)
      231 (69)38 (84)
      31 (2.2)0 (0)
      Total gestational sacs, n (%)0.06
      013 (29)22 (49)
      115 (33)16 (36)
      216 (36)7 (16)
      31 (2.2)0 (0)
      Donated oocytes, n (%)0.62
      Statistically significant.
      5–1022 (48.9)24 (53.3)
      11–2021 (46.7)20 (44.4)
      >212 (4.4)1 (2.2)
      Fertilization rate, n (%)0.13
      Statistically significant.
      < 607 (15.6)16 (35.6)
      60–7917 (37.8)16 (35.6)
      >8021 (46.7)13 (28.9)
      Mean, %74.868.6
      Final cycle outcomes, n (%)0.02
      Statistically significant.
      Biochemical pregnancy
      Early pregnancy loss after positive beta HCG test without evidence of intrauterine pregnancy on ultrasound.
      1 (2.2)7 (16)
      Ectopic pregnancy
      Positive beta HCG test with confirmed extrauterine pregnancy on ultrasound.
      1 (2.2)0 (0)
      Live birth
      Delivery of live infant at 24 weeks of gestation or greater.
      31 (69)19 (42)
      No pregnancy11 (24)15 (33)
      Spontaneous miscarriage
      Pregnancy loss after positive beta HCG test with confirmed intrauterine pregnancy on ultrasound at less than 24 weeks of gestation.
      1 (2.2)3 (6.7)
      Stillbirth
      Delivery of a demised infant at 24 weeks of gestation or greater.
      0 (0)1 (2.2)
      Values are reported as median (interquartile range) or n (%).
      a Early pregnancy loss after positive beta HCG test without evidence of intrauterine pregnancy on ultrasound.
      b Positive beta HCG test with confirmed extrauterine pregnancy on ultrasound.
      c Delivery of live infant at 24 weeks of gestation or greater.
      d Pregnancy loss after positive beta HCG test with confirmed intrauterine pregnancy on ultrasound at less than 24 weeks of gestation.
      e Delivery of a demised infant at 24 weeks of gestation or greater.
      f Statistically significant.
      Assisted reproductive technology outcomes are presented in Table 2. The implantation rate was 64% in the fresh sperm group and 36% in the frozen sperm group (P = 0.04). The positive pregnancy rate was 76% in the fresh sperm group and 67% in the frozen sperm group (P = 0.49). The delivery rate was 69% in the fresh sperm group and 44% in the frozen sperm group (P = 0.03). The rate of pregnancy loss was 33% in the frozen sperm group and 5.9% in the fresh sperm group (P = 0.01).
      TABLE 2ASSISTED REPRODUCTIVE TECHNOLOGY OUTCOMES BY TYPE OF EJACULATED SPERMATOZOA
      Spermatozoa
      Fresh (n = 45)Frozen (n = 45)P-value
      Implantation
      Calculated based on total number of gestational sacs seen over number of embryos transferred.
      , n (%)
      50/78 (64)30/83 (36)0.04
      Pregnancy
      Positive beta HCG test on cycle day 28 (serum concentration >5 mIU/ml).
      , n (%)
      34 (76)
      Includes one ectopic pregnancy.
      30 (67)0.49
      Pregnancy loss
      Includes biochemical pregnancy losses and clinical pregnancy losses before 24 weeks’ gestation.
      , n (%)
      2 (5.9)10 (33)0.01
      Statistically significant.
      Delivery, n (%)31 (69)20 (44)
      Includes one stillbirth.
      0.03
      Statistically significant.
      a Calculated based on total number of gestational sacs seen over number of embryos transferred.
      b Positive beta HCG test on cycle day 28 (serum concentration >5 mIU/ml).
      c Includes biochemical pregnancy losses and clinical pregnancy losses before 24 weeks’ gestation.
      d Includes one ectopic pregnancy.
      e Includes one stillbirth.
      f Statistically significant.
      The results of the mixed-effects logistic regression analysis are presented in Supplementary Tables 1–3. The adjusted odds of implantation was not significantly different in the frozen compared with the fresh sperm group when controlling for donor spermatozoa and gravidity (adjusted OR 0.5, 95% CI 0.12, 2.12). The adjusted odds of pregnancy was not significantly different in the frozen sperm group compared with the fresh sperm group when controlling for partner age (adjusted OR 0.67, 95% CI 0.20, 2.27). The adjusted odds of delivery was 67% lower for patients in the frozen sperm group when controlling for number of embryos transferred (adjusted OR 0.33, 95% CI 0.12, 0.89).

      Discussion

      In this retrospective study, we found that IVF-ICSI cycles using fresh ejaculated spermatozoa was associated with higher delivery rates and lower rates of pregnancy loss than cycles using frozen ejaculated spermatozoa. Although implantation and pregnancy rates seemed to be higher in the fresh ejaculated sperm group, these results did not reach statistical significance.
      To the best of our knowledge, no study has compared ART outcomes of fresh and frozen ejaculated spermatozoa using sibling oocytes in couples with non-male factor infertility. Sibling oocyte donor recipients receiving oocytes from the same donor stimulation transferred to two different recipients offers a unique ability to control for oocyte quality to the greatest degree possible. The use of sibling oocyte recipients also controls for the heterogeneity involved with various stimulation cycles and, therefore, allows a less biased comparison between the recipients. It is also important to note that no significant difference was found between the two recipients in recipient baseline demographics, body mass index, endometrial thickness, history of prior uterine surgery, number of embryos transferred and day of embryo transfer, thereby decreasing other biases that may affect endometrial receptivity. Additionally, mixed-effects logistic regression was carried out to account for the paired nature of the sibling oocytes.
      Cryopreservation of spermatozoa is widely used in IVF owing to its many benefits. It most importantly allows for the storage of spermatozoa (
      • Borges Edson
      • Rossi Lia Mara
      • Victor Locambo de Freitas Christiany
      • Guilherme Patrícia
      • Carvalho Tatiana
      • Bonetti S.
      • Iaconelli Assumpto
      • Pasqualotto Fabio Firmbach
      Fertilization and Pregnancy Outcome after Intracytoplasmic Injection with Fresh or Cryopreserved Ejaculated Spermatozoa.
      ), which creates more flexibility in IVF cycles, as sperm production and oocyte retrievals do not need to be coordinated (
      • Oates R.D.
      • Lobel S.M.
      • Harris D.H.
      • Pang S.
      • Burgess C.M.
      • Carson R.S.
      Efficacy of Intracytoplasmic Sperm Injection Using Intentionally Cryopreserved Epididymal Spermatozoa.
      ). It, therefore, avoids the pressure and potential delays in transfers if retrievals do not go as planned. It also allows for the more convenient use of donor spermatozoa and avoids patients with male factor infertility from undergoing multiple surgical procedures (
      • Kalsi Jas
      • Yau Thum M.
      • Muneer Asif
      • Pryor John
      • Abdullah Hossam
      • Minhas Suks
      Analysis of the Outcome of Intracytoplasmic Sperm Injection Using Fresh or Frozen Sperm.
      ). Cryopreservation of spermatozoa, however, can lead to decreased fertilization rates by causing damage to sperm DNA and acrosomal structure (Verheyen et al., 1997;
      • Chatterjee S.
      • Gagnon C.
      Production of Reactive Oxygen Species by Spermatozoa Undergoing Cooling, Freezing, and Thawing.
      ;
      • Thompson-Cree M.E.M.
      • McClure Neil
      • Donnelly Eilish T.
      • Steele Kristine E.
      • Lewis Sheena E.M.
      Effects of Cryopreservation on Testicular Sperm Nuclear DNA Fragmentation and Its Relationship with Assisted Conception Outcome Following ICSI with Testicular Spermatozoa.
      ) and by impairing sperm motility (
      • Donnelly E.T.
      • McClure N.
      • Lewis S.E.
      Cryopreservation of Human Semen and Prepared Sperm: Effects on Motility Parameters and DNA Integrity.
      ;
      • Donnelly E.T.
      • Steele E.K.
      • McClure N.
      • Lewis S.E.
      Assessment of DNA Integrity and Morphology of Ejaculated Spermatozoa from Fertile and Infertile Men before and after Cryopreservation.
      ). Although cryopreservation of spermatozoa has been associated with poorer outcomes in intrauterine inseminations, most studies have shown that it does not affect outcomes in IVF-ICSI cycles (
      • Nagy Z.
      • Liu J.
      • Cecile J.
      • Silber S.
      • Devroey P.
      • Van Steirteghem A.
      Using Ejaculated, Fresh, and Frozen-Thawed Epididymal and Testicular Spermatozoa Gives Rise to Comparable Results after Intracytoplasmic Sperm Injection.
      ;
      • Tournaye Herman
      • Merdad Talal
      • Silber Sherman
      • Joris Hubert
      • Verheyen Greta
      • Devroey Paul
      • Van Steirteghem André
      No Differences in Outcome after Intracytoplasmic Sperm Injection with Fresh or with Frozen–Thawed Epididymal Spermatozoa.
      ;
      • Cayan Selahittin
      • Lee Douglas
      • Conaghan Joseph
      • Givens Carolyn A.
      • Ryan Isabelle P.
      • Schriock Eldon D.
      • Turek Paul J.
      A Comparison of ICSI Outcomes with Fresh and Cryopreserved Epididymal Spermatozoa from the Same Couples.
      ;
      • Ulug Ulun
      • Bener Faruk
      • Karagenc Levent
      • Ciray Nadir
      • Bahceci Mustafa
      Outcomes in Couples Undergoing ICSI: Comparison between Fresh and Frozen–Thawed Surgically Retrieved Spermatozoa.
      ;
      • Kalsi Jas
      • Yau Thum M.
      • Muneer Asif
      • Pryor John
      • Abdullah Hossam
      • Minhas Suks
      Analysis of the Outcome of Intracytoplasmic Sperm Injection Using Fresh or Frozen Sperm.
      ;
      • Yu Zhe
      • Wei Zhewen
      • Yang Jun
      • Wang Tao
      • Jiang Hongyang
      • Li Hao
      • Tang Zhe
      • Wang Shaogang
      • Liu Jihong
      Comparison of Intracytoplasmic Sperm Injection Outcome with Fresh versus Frozen-Thawed Testicular Sperm in Men with Nonobstructive Azoospermia: A Systematic Review and Meta-Analysis.
      ). Cryopreservation of spermatozoa may actually be beneficial in IVF-ICSI cycles, as freezing of spermatozoa may select against defective spermatozoa and isolate a more optimal spermatozoa to be used for fertilization (
      • Tournaye Herman
      • Merdad Talal
      • Silber Sherman
      • Joris Hubert
      • Verheyen Greta
      • Devroey Paul
      • Van Steirteghem André
      No Differences in Outcome after Intracytoplasmic Sperm Injection with Fresh or with Frozen–Thawed Epididymal Spermatozoa.
      ).
      • Lara-Cerrillo Sandra
      • Ribas-Maynou Jordi
      • Rosado-Iglesias Candela
      • Lacruz-Ruiz Tania
      • Benet Jordi
      • García-Peiró Agustín
      Sperm Selection during ICSI Treatments Reduces Single- but Not Double-Strand DNA Break Values Compared to the Semen Sample.
      found that ICSI may select against spermatozoa with single-strand DNA breaks, although it may be less effective at identifying spermatozoa with double-strand DNA breaks.
      Studies that evaluated fresh and frozen spermatozoa in IVF-ICSI cycles have shown pregnancy outcomes to be similar; however, these studies have focused on couples with male factor infertility and surgically retrieved spermatozoa (
      • Nagy Z.
      • Liu J.
      • Cecile J.
      • Silber S.
      • Devroey P.
      • Van Steirteghem A.
      Using Ejaculated, Fresh, and Frozen-Thawed Epididymal and Testicular Spermatozoa Gives Rise to Comparable Results after Intracytoplasmic Sperm Injection.
      ;
      • Tournaye Herman
      • Merdad Talal
      • Silber Sherman
      • Joris Hubert
      • Verheyen Greta
      • Devroey Paul
      • Van Steirteghem André
      No Differences in Outcome after Intracytoplasmic Sperm Injection with Fresh or with Frozen–Thawed Epididymal Spermatozoa.
      ;
      • Cayan Selahittin
      • Lee Douglas
      • Conaghan Joseph
      • Givens Carolyn A.
      • Ryan Isabelle P.
      • Schriock Eldon D.
      • Turek Paul J.
      A Comparison of ICSI Outcomes with Fresh and Cryopreserved Epididymal Spermatozoa from the Same Couples.
      ;
      • Ulug Ulun
      • Bener Faruk
      • Karagenc Levent
      • Ciray Nadir
      • Bahceci Mustafa
      Outcomes in Couples Undergoing ICSI: Comparison between Fresh and Frozen–Thawed Surgically Retrieved Spermatozoa.
      ;
      • Kalsi Jas
      • Yau Thum M.
      • Muneer Asif
      • Pryor John
      • Abdullah Hossam
      • Minhas Suks
      Analysis of the Outcome of Intracytoplasmic Sperm Injection Using Fresh or Frozen Sperm.
      ;
      • Yu Zhe
      • Wei Zhewen
      • Yang Jun
      • Wang Tao
      • Jiang Hongyang
      • Li Hao
      • Tang Zhe
      • Wang Shaogang
      • Liu Jihong
      Comparison of Intracytoplasmic Sperm Injection Outcome with Fresh versus Frozen-Thawed Testicular Sperm in Men with Nonobstructive Azoospermia: A Systematic Review and Meta-Analysis.
      ). Only a few studies have compared fresh and frozen ejaculated spermatozoa in ICSI in couples with non-male factor infertility. As ICSI is now commonly used in clinical practice, understanding pregnancy outcomes in patients using ICSI with non-male factor infertility is necessary.
      In the literature review, only one other study compared pregnancy outcomes between fresh and frozen ejaculated spermatozoa in IVF-ICSI cycles in patients without male factor infertility. In their retrospective study,
      • Borges Edson
      • Rossi Lia Mara
      • Victor Locambo de Freitas Christiany
      • Guilherme Patrícia
      • Carvalho Tatiana
      • Bonetti S.
      • Iaconelli Assumpto
      • Pasqualotto Fabio Firmbach
      Fertilization and Pregnancy Outcome after Intracytoplasmic Injection with Fresh or Cryopreserved Ejaculated Spermatozoa.
      found that fresh and frozen ejaculated spermatozoa resulted in similar fertilization, implantation and pregnancy rates in patients with normozoospermia.
      • Borges Edson
      • Rossi Lia Mara
      • Victor Locambo de Freitas Christiany
      • Guilherme Patrícia
      • Carvalho Tatiana
      • Bonetti S.
      • Iaconelli Assumpto
      • Pasqualotto Fabio Firmbach
      Fertilization and Pregnancy Outcome after Intracytoplasmic Injection with Fresh or Cryopreserved Ejaculated Spermatozoa.
      , however, did not use donor oocytes and was limited by only 44 patients with normal semen analyses. Although our study also resulted in similar fertilization, implantation and pregnancy rates, our outcomes favoured the fresh sperm cohort, which ultimately had significantly higher delivery rates and lower rates of pregnancy loss than the frozen ejaculated sperm group. Given the lower delivery rate in the frozen sperm cohort, our results suggest that freezing spermatozoa compromises sperm quality and that ICSI may not overcome this reduced reproductive capacity of frozen spermatozoa. Although DNA damage caused by cryopreservation may have contributed to the difference in outcomes, because sperm characteristics were not specifically evaluated, it is unknown how cryopreservation decreased sperm quality and contributed to the difference in outcomes.
      The present study only included patients with non-male factor infertility; however, it is important to note that only the frozen ejaculated sperm group contained patients using donor spermatozoa. As sperm donors have undergone a thorough selection process, it is necessary to address that these samples did not result in improved outcomes compared with the fresh ejaculated sperm group.
      Limitations of the study include a small sample size, which is inherent in a study using sibling oocyte recipients, as the recipients must have discrepant sources of spermatozoa to be compared. As a retrospective study based on chart review, the study is limited in access to data dependent on provider documentation and patient follow-up. Although the present study focused on pregnancy outcomes, embryo quality specifically was not evaluated. All semen analyses were normal, as patients with male factor infertility and suboptimal semen analyses were excluded; however, sperm characteristics were not specifically evaluated. Future research should further investigate semen characteristics and its association with pregnancy outcomes. Additionally, owing to limitations in data availability, cumulative delivery rates could not be assessed. Another limitation of the study is that we included both frozen donor spermatozoa and partner frozen spermatozoa, and it is unknown whether the same cryopreservation methods used in our centre are the same as those used across the various sperm banks nationally. As such, the effect of varying cryopreservatives could not be controlled for.
      In conclusion, in this idealized model that controls for oocyte quality to the greatest degree possible, we found that IVF-ICSI cycles using frozen ejaculated spermatozoa had worse pregnancy outcomes through reduced delivery rates and higher pregnancy loss rates than those using fresh ejaculated sperm. Cryopreservation of spermatozoa allows for easier coordination of cycles; however, our results suggest that fresh ejaculated spermatozoa may be preferable in IVF-ICSI cycles in patients with non-male factor infertility.

      Uncited Reference

      • Verheyen G.
      • Nagy Z.
      • Joris H.
      • De Croo I.
      • Tournaye H.
      • Van Steirteghem A.
      Quality of Frozen-Thawed Testicular Sperm and Its Preclinical Use for Intracytoplasmic Sperm Injection into in Vitro-Matured Germinal-Vesicle Stage Oocytes.

      Acknowledgements

      Anjile An, MPH, was partially supported by the following grant: Clinical and Translational Science Center at Weill Cornell Medical College (1-UL1-TR002384-01). Preliminary data were presented at the 2020 ASRM Annual Meeting.

      Appendix. Supplementary materials

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      Biography

      Dr Steven Spandorfer is Associate Professor of Obstetrics and Gynecology and Reproductive Medicine at the Ronald O Perelman and Claudia Cohen Center for Reproductive Medicine at Weill Cornell Medical College. A renowned reproductive medicine specialist, he is board certified in both obstetrics and gynaecology and reproductive endocrinology and infertility.
      Key message
      IVF and intracytoplasmic sperm injection (ICSI) cycles using frozen ejaculated spermatozoa results in poorer pregnancy outcomes compared with cycles using fresh ejaculated spermatozoa in couples with non-male factor infertility. Our study suggests that fresh ejaculated spermatozoa may be preferable in IVF-ICSI cycles in patients with non-male factor infertility.